Ovarian cancer-associated antibodies and kits

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, particularly ovarian cancer, are disclosed. Illustrative compositions comprise one or more ovarian tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly ovarian cancer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to therapy and diagnosis ofcancer, such as ovarian cancer. The invention is more specificallyrelated to polypeptides, comprising at least a portion of an ovariantumor protein, and to polynucleotides encoding such polypeptides. Suchpolypeptides and polynucleotides are useful in pharmaceuticalcompositions, e.g., vaccines, and other compositions for the diagnosisand treatment of ovarian cancer.

2. Description of the Related Art

Cancer is a significant health problem throughout the world. Althoughadvances have been made in detection and therapy of cancer, no vaccineor other universally successful method for prevention and/or treatmentis currently available. Current therapies, which are generally based ona combination of chemotherapy or surgery and radiation, continue toprove inadequate in many patients.

Ovarian cancer is a significant health problem for women in the UnitedStates and throughout the world. Although advances have been made indetection and therapy of this cancer, no vaccine or other universallysuccessful method for prevention or treatment is currently available.Management of the disease currently relies on a combination of earlydiagnosis and aggressive treatment, which may include one or more of avariety of treatments such as surgery, radiotherapy, chemotherapy andhormone therapy. The course of treatment for a particular cancer isoften selected based on a variety of prognostic parameters, including ananalysis of specific tumor markers. However, the use of establishedmarkers often leads to a result that is difficult to interpret, and highmortality continues to be observed in many cancer patients.

Immunotherapies have the potential to substantially improve cancertreatment and survival. Such therapies may involve the generation orenhancement of an immune response to an ovarian carcinoma antigen.However, to date, relatively few ovarian carcinoma antigens are knownand the generation of an immune response against such antigens has notbeen shown to be therapeutically beneficial.

Accordingly, there is a need in the art for improved methods foridentifying ovarian tumor antigens and for using such antigens in thetherapy of ovarian cancer. The present invention fulfills these needsand further provides other related advantages.

In spite of considerable research into therapies for these and othercancers, ovarian cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers. The present inventionfulfills these needs and further provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

(a) sequences provided in SEQ ID NOs: 1-311, 313-387, 391, 457, 460-477,512-570 and 619-622;

(b) complements of the sequences provided in SEQ ID NOs: 1-311, 313-387,391, 457, 460-477, 512-570 and 619-622;

(c) sequences consisting of at least 20, 25, 30, 35, 40, 45, 50, 75 and100 contiguous residues of a sequence provided in SEQ ID NOs: 1-311,313-387, 391, 457, 460-477, 512-570 and 619-622;

(d) sequences that hybridize to a sequence provided in SEQ ID NOs:1-311, 313-387, 391, 457, 460-477, 512-570 and 619-622, under moderateor highly stringent conditions;

(e) sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identity to a sequence of SEQ ID NOs: 1-311, 313-387, 391, 457,460-477, 512-570 and 619-622;

(f) degenerate variants of a sequence provided in SEQ ID NOs: 1-311,313-387, 391, 457, 460-477, 512-570 and 619-622.

In one preferred embodiment, the polynucleotide compositions of theinvention are expressed in at least about 20%, more preferably in atleast about 30%, and most preferably in at least about 50% of ovariantumors samples tested, at a level that is at least about 2-fold,preferably at least about 5-fold, and most preferably at least about10-fold higher than that for normal tissues.

The present invention, in another aspect, provides polypeptidecompositions comprising an amino acid sequence that is encoded by apolynucleotide sequence described above.

The present invention further provides polypeptide compositionscomprising an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NOs: 312, 388-389, 392-455, 458-459,478-511, 571-618 and 623-624.

In certain preferred embodiments, the polypeptides and/orpolynucleotides of the present invention are immunogenic, i.e., they arecapable of eliciting an immune response, particularly a humoral and/orcellular immune response, as further described herein.

The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide and/or polynucleotidesequences, wherein the fragments, variants and/or derivatives preferablyhave a level of immunogenic activity of at least about 50%, preferablyat least about 70% and more preferably at least about 90% of the levelof immunogenic activity of a polypeptide sequence set forth in SEQ IDNOs: 312, 388-389, 392-455,458-459, 478-511, and 571-618 and 623-624 ora polypeptide sequence encoded by a polynucleotide sequence set forth inSEQ ID NOs: 1-311, 313-387, 391, 457, 460-477, 512-570 and 619-622.

The present invention further provides polynucleotides that encode apolypeptide described above, expression vectors comprising suchpolynucleotides and host cells transformed or transfected with suchexpression vectors.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, the pharmaceuticalcompositions, e.g., vaccine compositions, are provided for prophylacticor therapeutic applications. Such compositions generally comprise animmunogenic polypeptide or polynucleotide of the invention and animmunostimulant, such as an adjuvant.

The present invention further provides pharmaceutical compositions thatcomprise: (a) an antibody or antigen-binding fragment thereof thatspecifically binds to a polypeptide of the present invention, or afragment thereof; and (b) a physiologically acceptable carrier.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Illustrative antigen presenting cells includedendritic cells, macrophages, monocytes, fibroblasts and B cells.

Within related aspects, pharmaceutical compositions are provided thatcomprise: (a) an antigen presenting cell that expresses a polypeptide asdescribed above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

Within further aspects, the present invention provides methods forstimulating an immune response in a patient, preferably a T cellresponse in a human patient, comprising administering a pharmaceuticalcomposition described herein. The patient may be afflicted with ovariancancer, in which case the methods provide treatment for the disease, orpatient considered at risk for such a disease may be treatedprophylactically.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition as recitedabove. The patient may be afflicted with ovarian cancer, in which casethe methods provide treatment for the disease, or patient considered atrisk for such a disease may be treated prophylactically.

The present invention further provides, within other aspects, methodsfor removing tumor cells from a biological sample, comprising contactinga biological sample with T cells that specifically react with apolypeptide of the present invention, wherein the step of contacting isperformed under conditions and for a time sufficient to permit theremoval of cells expressing the protein from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a polypeptide of the presentinvention, comprising contacting T cells with one or more of: (i) apolypeptide as described above; (ii) a polynucleotide encoding such apolypeptide; and/or (iii) an antigen presenting cell that expresses sucha polypeptide; under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells. Isolated T cell populationscomprising T cells prepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofpolypeptide disclosed herein; (ii) a polynucleotide encoding such apolypeptide; and (iii) an antigen-presenting cell that expressed such apolypeptide; and (b) administering to the patient an effective amount ofthe proliferated T cells, and thereby inhibiting the development of acancer in the patient. Proliferated cells may, but need not, be clonedprior to administration to the patient.

Within further aspects, the present invention provides methods fordetermining the presence or absence of a cancer, preferably an ovariancancer, in a patient comprising: (a) contacting a biological sampleobtained from a patient with a binding agent that binds to a polypeptideas recited above; (b) detecting in the sample an amount of polypeptidethat binds to the binding agent; and (c) comparing the amount ofpolypeptide with a predetermined cut-off value, and therefromdetermining the presence or absence of a cancer in the patient. Withinpreferred embodiments, the binding agent is an antibody, more preferablya monoclonal antibody.

The present invention also provides, within other aspects, methods formonitoring the progression of a cancer in a patient. Such methodscomprise the steps of: (a) contacting a biological sample obtained froma patient at a first point in time with a binding agent that binds to apolypeptide as recited above; (b) detecting in the sample an amount ofpolypeptide that binds to the binding agent; (c) repeating steps (a) and(b) using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polypeptide detected instep (c) with the amount detected in step (b) and therefrom monitoringthe progression of the cancer in the patient.

The present invention further provides, within other aspects, methodsfor determining the presence or absence of a cancer in a patient,comprising the steps of: (a) contacting a biological sample, e.g., tumorsample, serum sample, etc., obtained from a patient with anoligonucleotide that hybridizes to a polynucleotide that encodes apolypeptide of the present invention; (b) detecting in the sample alevel of a polynucleotide, preferably mRNA, that hybridizes to theoligonucleotide; and (c) comparing the level of polynucleotide thathybridizes to the oligonucleotide with a predetermined cut-off value,and therefrom determining the presence or absence of a cancer in thepatient. Within certain embodiments, the amount of mRNA is detected viapolymerase chain reaction using, for example, at least oneoligonucleotide primer that hybridizes to a polynucleotide encoding apolypeptide as recited above, or a complement of such a polynucleotide.Within other embodiments, the amount of mRNA is detected using ahybridization technique, employing an oligonucleotide probe thathybridizes to a polynucleotide that encodes a polypeptide as recitedabove, or a complement of such a polynucleotide.

In related aspects, methods are provided for monitoring the progressionof a cancer in a patient, comprising the steps of: (a) contacting abiological sample obtained from a patient with an oligonucleotide thathybridizes to a polynucleotide that encodes a polypeptide of the presentinvention; (b) detecting in the sample an amount of a polynucleotidethat hybridizes to the oligonucleotide; (c) repeating steps (a) and (b)using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polynucleotide detectedin step (c) with the amount detected in step (b) and therefrommonitoring the progression of the cancer in the patient.

Within further aspects, the present invention provides antibodies, suchas monoclonal antibodies, that bind to a polypeptide as described above,as well as diagnostic kits comprising such antibodies. Diagnostic kitscomprising one or more oligonucleotide probes or primers as describedabove are also provided.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-1S (SEQ ID NO:1-71) depict partial sequences of polynucleotidesencoding representative secreted ovarian carcinoma antigens.

FIGS. 2A-2C depict full insert sequences for three of the clones ofFIG. 1. FIG. 2A shows the sequence designated O7E (11731; SEQ ID NO:72),FIG. 2B shows the sequence designated O9E (11785; SEQ ID NO:73) and FIG.2C shows the sequence designated O8E (13695; SEQ ID NO:74).

FIG. 3 presents results of microarray expression analysis of the ovariancarcinoma sequence designated O8E.

FIG. 4 presents a partial sequence of a polynucleotide (designated 3g;SEQ ID NO:75) encoding an ovarian carcinoma sequence that is a splicefusion between the human T-cell leukemia virus type I oncoprotein TAXand osteonectin.

FIG. 5 presents the ovarian carcinoma polynucleotide designated 3f (SEQID NO:76).

FIG. 6 presents the ovarian carcinoma polynucleotide designated 6b (SEQID NO:77).

FIGS. 7A and 7B present the ovarian carcinoma polynucleotides designated8e (SEQ ID NO:78) and 8h (SEQ ID NO:79).

FIG. 8 presents the ovarian carcinoma polynucleotide designated 12c (SEQID NO:80).

FIG. 9 presents the ovarian carcinoma polynucleotide designated 12h (SEQID NO:81).

FIG. 10 depicts results of microarray expression analysis of the ovariancarcinoma sequence designated 3f.

FIG. 11 depicts results of microarray expression analysis of the ovariancarcinoma sequence designated 6b.

FIG. 12 depicts results of microarray expression analysis of the ovariancarcinoma sequence designated 8e.

FIG. 13 depicts results of microarray expression analysis of the ovariancarcinoma sequence designated 12c.

FIG. 14 depicts results of microarray expression analysis of the ovariancarcinoma sequence designated 12h.

FIGS. 15A-15E2 depict partial sequences of additional polynucleotidesencoding representative secreted ovarian carcinoma antigens (SEQ IDNO:82-310).

FIG. 16 is a diagram illustrating the location of various partial O8Esequences within the full length sequence.

FIG. 17 is a graph illustrating the results of epitope mapping studieson O8E protein.

FIG. 18 is graph of a fluorescence activated cell sorting (FACS)analysis of O8E cell surface expression.

FIG. 19 is graph of a FACS analysis of O8E cell surface expression.

FIG. 20 shows FACS analysis results for O8E transfected HEK293 cellsdemonstrating cell surface expression of O8E.

FIG. 21 shows FACS analysis results for SKBR3 breast tumor cellsdemonstrating cell surface expression of O8E.

FIG. 22 shows O8E expression in HEK 293 cells. The cells were probedwith anti-O8E rabbit polyclonal antisera #2333L.

FIG. 23 shows the ELISA analysis of anti-O8E rabbit sera.

FIG. 24 shows the ELISA analysis of affinity purified rabbit anti-08Epolyclonal antibody.

FIG. 25 is a graph determining antibody internalization of anti-O8E mAbshowing that mAbs against amino acids 61-80 induces ligandinternalization.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NO:1-71 are ovarian carcinoma antigen polynucleotides shown inFIGS. 1A-1S.

SEQ ID NO:72-74 are ovarian carcinoma antigen polynucleotides shown inFIGS. 2A-2C.

SEQ ID NO:75 is the ovarian carcinoma polynucleotide 3g (FIG. 4).

SEQ ID NO:76 is the ovarian carcinoma polynucleotide 3f (FIG. 5).

SEQ ID NO:77 is the ovarian carcinoma polynucleotide 6b (FIG. 6).

SEQ ID NO:78 is the ovarian carcinoma polynucleotide 8e (FIG. 7A).

SEQ ID NO:79 is the ovarian carcinoma polynucleotide 8h (FIG. 7B).

SEQ ID NO:80 is the ovarian carcinoma polynucleotide 12e (FIG. 8).

SEQ ID NO:81 is the ovarian carcinoma polynucleotide 12h (FIG. 9).

SEQ ID NO:82-310 are ovarian carcinoma antigen polynucleotides shown inFIGS. 15A-15EEE.

SEQ ID NO:311 is a full length sequence of ovarian carcinomapolynucleotide O772P.

SEQ ID NO:312 is the O772P amino acid sequence.

SEQ ID NO:313-384 are ovarian carcinoma antigen polynucleotides.

SEQ ID NO:385 represents the cDNA sequence of a form of the clone O772P,designated 21013.

SEQ ID NO:386 represents the cDNA sequence of a form of the clone O772P,designated 21003.

SEQ ID NO:387 represents the cDNA sequence of a form of the clone O772P,designated 21008.

SEQ ID NOs:388 is the amino acid sequence corresponding to SEQ IDNO:385.

SEQ ID NOs:389 is the amino acid sequence corresponding to SEQ IDNO:386.

SEQ ID NOs:390 is the amino acid sequence corresponding to SEQ IDNO:387.

SEQ ID NO:391 is a full length sequence of ovarian carcinomapolynucleotide O8E.

SEQ ID NO:392-393 are protein sequences encoded by O8E.

SEQ ID NO:394-415 are peptide sequences corresponding to the O8Eantibody epitopes.

SEQ ID NO:416-435 are potential HLA-A2 10-mer binding peptides predictedusing the full length open-reading frame from O8E.

SEQ ID NO:436-455 are potential HLA-A2 9-mer binding peptides predictedusing the full length open-reading frame from O8E.

SEQ ID NO:456 is a truncated nucleotide sequence of the full lengthGenbank sequence showing homology to O772P

SEQ ID NO:457 is the full length Genbank sequence showing significanthomology to O772P

SEQ ID NO:458 is a protein encoding a truncated version of the fulllength Genbank sequence showing homology to O772P

SEQ ID NO:459 is the full length protein sequence from Genbank showingsignificant homology to the protein sequence for O772P

SEQ ID NO:460 encodes a unique N-terminal portion of O772P contained inresidues 1-70.

SEQ ID NO:461 contains unique sequence and encodes residues 1-313 of SEQID NO: 456.

SEQ ID NO:462 is the hypothetical sequence for clone O772P.

SEQ ID NO:463 is the cDNA sequence for clone FLJ14303.

SEQ ID NO:464 is a partial cDNA sequence for clone O772P.

SEQ ID NO:465 is a partial cDNA sequence for clone O772P.

SEQ ID NO:466 is a partial cDNA sequence for clone O772P.

SEQ ID NO:467 is a partial cDNA sequence for clone O772P.

SEQ ID NO:468 is a partial cDNA sequence for clone O772P.

SEQ ID NO:469 is a partial cDNA sequence for clone O772P.

SEQ ID NO:470 is a partial cDNA sequence for clone O772P.

SEQ ID NO:471 is a partial cDNA sequence for clone O772P.

SEQ ID NO:472 is a partial cDNA sequence for clone O772P.

SEQ ID NO:473 is a partial cDNA sequence for clone O772P.

SEQ ID NO:474 is a partial cDNA sequence for clone O772P.

SEQ ID NO:475 is a partial cDNA sequence for clone O772P.

SEQ ID NO:476 is a partial cDNA sequence for clone O772P.

SEQ ID NO:477 represents the novel 5′-end of the ovarian tumor antigenO772P.

SEQ ID NO:478 is the amino acid sequence encoded by SEQ ID NO:462.

SEQ ID NO:479 is the amino acid sequence encoded by SEQ ID NO:463.

SEQ ID NO:480 is a partial amino acid sequence encoded by SEQ ID NO:472.

SEQ ID NO:481 is a partial amino acid sequence encoded by a possibleopen reading frame of SEQ ID NO:471.

SEQ ID NO:482 is a partial amino acid sequence encoded by a secondpossible open reading frame of SEQ ID NO:471.

SEQ ID NO:483 is a partial amino acid sequence encoded by SEQ ID NO:467.

SEQ ID NO:484 is a partial amino acid sequence encoded by a possibleopen reading frame of SEQ ID NO:466.

SEQ ID NO:485 is a partial amino acid sequence encoded by a secondpossible open reading frame of SEQ ID NO:466.

SEQ ID NO:486 is a partial amino acid sequence encoded by SEQ ID NO:465.

SEQ ID NO:487 is a partial amino acid sequence encoded by SEQ ID NO:464.

SEQ ID NO:488 represents the extracellular, transmembrane andcytoplasmic regions of O772P.

SEQ ID NO:489 represents the predicted extracellular domain of O772P.

SEQ ID NO:490 represents the amino acid sequence of peptide #2 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:491 represents the amino acid sequence of peptide #6 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:492 represents the amino acid sequence of peptide #7 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:493 represents the amino acid sequence of peptide #8 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:494 represents the amino acid sequence of peptide #9 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:495 represents the amino acid sequence of peptide #11 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:496 represents the amino acid sequence of peptide #13 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:497 represents the amino acid sequence of peptide #22 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:498 represents the amino acid sequence of peptide #24 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:499 represents the amino acid sequence of peptide #27 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:500 represents the amino acid sequence of peptide #40 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:501 represents the amino acid sequence of peptide #41 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:502 represents the amino acid sequence of peptide #47 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:503 represents the amino acid sequence of peptide #50 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:504 represents the amino acid sequence of peptide #51 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:505 represents the amino acid sequence of peptide #52 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:506 represents the amino acid sequence of peptide #53 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:507 represents the amino acid sequence of peptide #58 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:508 represents the amino acid sequence of peptide #59 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:509 represents the amino acid sequence of peptide #60 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:510 represents the amino acid sequence of peptide #61 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:511 represents the amino acid sequence of peptide #71 whichcorresponds to an O772P specific antibody epitope.

SEQ ID NO:512 (O772P repeat1) represents an example of a cDNA sequencecorresponding to repeat number 21 from the 5′ variable region of O772P.

SEQ ID NO:513 (O772P repeat2) represents an example of a cDNA sequencecorresponding to repeat number 20 from the 5′ variable region of O772P.

SEQ ID NO:514 (O772P repeat3) represents an example of a cDNA sequencecorresponding to repeat number 19 from the 5′ variable region of O772P.

SEQ ID NO:515 (O772P repeat4) represents an example of a cDNA sequencecorresponding to repeat number 18 from the 5′ variable region of O772P.

SEQ ID NO:516 (O772P repeat5) represents an example of a cDNA sequencecorresponding to repeat number 17 from the 5′ variable region of O772P.

SEQ ID NO:517 (HB repeat1) represents an example of a cDNA sequencecorresponding to repeat number 21 from the 5′ variable region of O772P.

SEQ ID NO:518 (HB repeat2) represents an example of a cDNA sequencecorresponding to repeat number 20 from the 5′ variable region of O772P.

SEQ ID NO:519 (HB repeat3) represents an example of a cDNA sequencecorresponding to repeat number 19 from the 5′ variable region of O772P.

SEQ ID NO:520 (HB repeat4) represents an example of a cDNA sequencecorresponding to repeat number 18 from the 5′ variable region of O772P.

SEQ ID NO:521 (HB repeat5) represents an example of a cDNA sequencecorresponding to repeat number 17 from the 5′ variable region of O772P.

SEQ ID NO:522 (HB repeat6 5′-end) represents an example of a cDNAsequence corresponding to repeat number 16 from the 5′ variable regionof O772P.

SEQ ID NO:523 (1043400.1 repeat1) represents an example of a cDNAsequence corresponding to repeat number 9 from the 5′ variable region ofO772P.

SEQ ID NO:524 (1043400.1 repeat2) represents an example of a cDNAsequence corresponding to repeat number 10 from the 5′ variable regionof O772P.

SEQ ID NO:525 (1043400.1 repeat3) represents an example of a cDNAsequence corresponding to repeat No. 10/11 from the 5′ variable regionof O772P.

SEQ ID NO:526 (1043400.1 repeat4) represents an example of a cDNAsequence corresponding to repeat number 11 from the 5′ variable regionof O772P.

SEQ ID NO:527 (1043400.1 repeat5) represents an example of a cDNAsequence corresponding to repeat number 14 from the 5′ variable regionof O772P.

SEQ ID NO:528 (1043400.1 repeat6) represents an example of a cDNAsequence corresponding to repeat number 17 from the 5′ variable regionof O772P.

SEQ ID NO:529 (1043400.3 repeat1) represents an example of a cDNAsequence corresponding to repeat number 20 from the 5′ variable regionof O772P.

SEQ ID NO:530 (1043400.3 repeat2) represents an example of a cDNAsequence corresponding to repeat number 21 from the 5′ variable regionof O772P.

SEQ ID NO:531 (1043400.5 repeat1) represents an example of a cDNAsequence corresponding to repeat number 8 from the 5′ variable region ofO772P.

SEQ ID NO:532 (1043400.5 repeat2) represents an example of a cDNAsequence corresponding to repeat number 9 from the 5′ variable region ofO772P, in addition containing intron sequence.

SEQ ID NO:533 (1043400.5 repeat2) represents an example of a cDNAsequence corresponding to repeat number 9 from the 5′ variable region ofO772P.

SEQ ID NO:534 (1043400.8 repeat1) represents an example of a cDNAsequence corresponding to repeat number 17 from the 5′ variable regionof O772P.

SEQ ID NO:535 (1043400.8 repeat2) represents an example of a cDNAsequence corresponding to repeat number 18 from the 5′ variable regionof O772P.

SEQ ID NO:536 (1043400.8 repeat3) represents an example of a cDNAsequence corresponding to repeat number 19 from the 5′ variable regionof O772P.

SEQ ID NO:537 (1043400.9 repeat1) represents an example of a cDNAsequence corresponding to repeat number 4 from the 5′ variable region ofO772P.

SEQ ID NO:538 (1043400.9 repeat2) represents an example of a cDNAsequence corresponding to repeat number 5 from the 5′ variable region ofO772P.

SEQ ID NO:539 (1043400.9 repeat3) represents an example of a cDNAsequence corresponding to repeat number 7 from the 5′ variable region ofO772P.

SEQ ID NO:540 (1043400.9 repeat4) represents an example of a cDNAsequence corresponding to repeat number 8 from the 5′ variable region ofO772P.

SEQ ID NO:541 (1043400.11 repeat1) represents an example of a cDNAsequence corresponding to repeat number 1 from the 5′ variable region ofO772P.

SEQ ID NO:542 (1043400.11 repeat2) represents an example of a cDNAsequence corresponding to repeat number 2 from the 5′ variable region ofO772P.

SEQ ID NO:543 (1043400.11 repeat3) represents an example of a cDNAsequence corresponding to repeat number 3 from the 5′ variable region ofO772P.

SEQ ID NO:544 (1043400.11 repeat4) represents an example of a cDNAsequence corresponding to repeat number 11 from the 5′ variable regionof O772P.

SEQ ID NO:545 (1043400.11 repeat5) represents an example of a cDNAsequence corresponding to repeat number 12 from the 5′ variable regionof O772P.

SEQ ID NO:546 (1043400.12 repeat1) represents an example of a cDNAsequence corresponding to repeat number 20 from the 5′ variable regionof O772P.

SEQ ID NO:547 (PB repeata) represents an example of a cDNA sequencecorresponding to repeat number 1 from the 5′ variable region of O772P.

SEQ ID NO:548 (PB repeatB) represents an example of a cDNA sequencecorresponding to repeat number 2 from the 5′ variable region of O772P.

SEQ ID NO:549 (PB repeatE) represents an example of a cDNA sequencecorresponding to repeat number 3 from the 5′ variable region of O772P.

SEQ ID NO:550 (PB repeatG) represents an example of a cDNA sequencecorresponding to repeat number 4 from the 5′ variable region of O772P.

SEQ ID NO:551 (PB repeatC) represents an example of a cDNA sequencecorresponding to repeat number 4 from the 5′ variable region of O772P.

SEQ ID NO:552 (PB repeatH) represents an example of a cDNA sequencecorresponding to repeat number 6 from the 5′ variable region of O772P.

SEQ ID NO:553 (PB repeatj) represents an example of a cDNA sequencecorresponding to repeat number 7 from the 5′ variable region of O772P.

SEQ ID NO:554 (PB repeatk) represents an example of a cDNA sequencecorresponding to repeat number 8 from the 5′ variable region of O772P.

SEQ ID NO:555 (PB repeatD) represents an example of a cDNA sequencecorresponding to repeat number 9 from the 5′ variable region of O772P.

SEQ ID NO:556 (PB repeatI) represents an example of a cDNA sequencecorresponding to repeat number 10 from the 5′ variable region of O772P.

SEQ ID NO:557 (PB repeatM) represents an example of a cDNA sequencecorresponding to repeat number 11 from the 5′ variable region of O772P.

SEQ ID NO:558 (PB repeat9) represents an example of a cDNA sequencecorresponding to repeat number 12 from the 5′ variable region of O772P.

SEQ ID NO:559 (PB repeat8.5) represents an example of a cDNA sequencecorresponding to repeat number 13 from the 5′ variable region of O772P.

SEQ ID NO:560 (PB repeat8) represents an example of a cDNA sequencecorresponding to repeat number 14 from the 5′ variable region of O772P.

SEQ ID NO:561 (PB repeat7) represents an example of a cDNA sequencecorresponding to repeat number 15 from the 5′ variable region of O772P.

SEQ ID NO:562 (PB repeat6) represents an example of a cDNA sequencecorresponding to repeat number 16 from the 5′ variable region of O772P.

SEQ ID NO:563 (PB repeat5) represents an example of a cDNA sequencecorresponding to repeat number 17 from the 5′ variable region of O772P.

SEQ ID NO:564 (PB repeat4) represents an example of a cDNA sequencecorresponding to repeat number 18 from the 5′ variable region of O772P.

SEQ ID NO:565 (PB repeat3) represents an example of a cDNA sequencecorresponding to repeat number 19 from the 5′ variable region of O772P.

SEQ ID NO:566 (PB repeat2) represents an example of a cDNA sequencecorresponding to repeat number 20 from the 5′ variable region of O772P.

SEQ ID NO:567 (PB repeat1) represents an example of a cDNA sequencecorresponding to repeat number 21 from the 5′ variable region of O772P.

SEQ ID NO:568 represents the cDNA sequence form the 3′ constant region.

SEQ ID NO:569 represents a cDNA sequence containing the consensussequences of the 21 repeats, the 3′ constant region and the 3′untranslated region.

SEQ ID NO:570 represents the cDNA sequence of the consensus repeatsequence.

SEQ ID NO:571 represents the consensus amino acid sequence of onepotential open reading frame of repeat number 1 from the 5′ variableregion of O772P.

SEQ ID NO:572 represents the consensus amino acid sequence of a secondpotential open reading frame of repeat number 1 from the 5′ variableregion of O772P.

SEQ ID NO:573 represents the consensus amino acid sequence of a thirdpotential open reading frame of repeat number 1 from the 5′ variableregion of O772P.

SEQ ID NO:574 represents the consensus amino acid sequence of repeatnumber 2 from the 5′ variable region of O772P.

SEQ ID NO:575 represents the consensus amino acid sequence of repeatnumber 3 from the 5′ variable region of O772P.

SEQ ID NO:576 represents the consensus amino acid sequence of repeatnumber 4 from the 5′ variable region of O772P.

SEQ ID NO:577 represents the consensus amino acid sequence of repeatnumber 5 from the 5′ variable region of O772P.

SEQ ID NO:578 represents the consensus amino acid sequence of repeatnumber 6 from the 5′ variable region of O772P.

SEQ ID NO:579 represents the consensus amino acid sequence of repeatnumber 7 from the 5′ variable region of O772P.

SEQ ID NO:580 represents the consensus amino acid sequence of repeatnumber 8 from the 5′ variable region of O772P.

SEQ ID NO:581 represents the consensus amino acid sequence of repeatnumber 9 from the 5′ variable region of O772P.

SEQ ID NO:582 represents the consensus amino acid sequence of repeatnumber 10 from the 5′ variable region of O772P.

SEQ ID NO:583 represents the consensus amino acid sequence of repeatnumber 11 from the 5′ variable region of O772P.

SEQ ID NO:584 represents the consensus amino acid sequence of repeatnumber 12 from the 5′ variable region of O772P.

SEQ ID NO:585 represents the consensus amino acid sequence of repeatnumber 13 from the 5′ variable region of O772P.

SEQ ID NO:586 represents the consensus amino acid sequence of repeatnumber 14 from the 5′ variable region of O772P.

SEQ ID NO:587 represents the consensus amino acid sequence of repeatnumber 15 from the 5′ variable region of O772P.

SEQ ID NO:588 represents the consensus amino acid sequence of repeatnumber 16 from the 5′ variable region of O772P.

SEQ ID NO:589 represents the consensus amino acid sequence of repeatnumber 17 from the 5′ variable region of O772P.

SEQ ID NO:590 represents the consensus amino acid sequence of repeatnumber 18 from the 5′ variable region of O772P.

SEQ ID NO:591 represents the consensus amino acid sequence of repeatnumber 19 from the 5′ variable region of O772P.

SEQ ID NO:592 represents the consensus amino acid sequence of repeatnumber 20 from the 5′ variable region of O772P.

SEQ ID NO:593 represents the consensus amino acid sequence of repeatnumber 21 from the 5′ variable region of O772P.

SEQ ID NO:594 represents the amino acid sequence of the 3′ constantregion.

SEQ ID NO:595 represents an amino acid sequence containing the consensussequences of the 21 repeats and the 3′ constant region.

SEQ ID NO:596 represents the amino acid sequence of the consensus repeatsequence.

SEQ ID NO:597 represents the amino acid sequence for Peptide #1, a30-mer peptide that corresponds to the predicted extracellular domain ofO772P.

SEQ ID NO:598 represents the amino acid sequence for Peptide #2, a30-mer peptide that corresponds to the predicted extracellular domain ofO772P.

SEQ ID NO:599 represents the amino acid sequence for Peptide #3, a30-mer peptide that corresponds to the predicted extracellular domain ofO772P.

SEQ ID NO:600 represents the amino acid sequence of Peptide #1 from O8E,which corresponds to amino acids 1-20.

SEQ ID NO:601 represents the amino acid sequence of Peptide #2 from O8E,which corresponds to amino acids 16-35.

SEQ ID NO:602 represents the amino acid sequence of Peptide #3 from O8E,which corresponds to amino acids 31-50.

SEQ ID NO:603 represents the amino acid sequence of Peptide #4 from O8E,which corresponds to amino acids 46-65.

SEQ ID NO:604 represents the amino acid sequence of Peptide #5 from O8E,which corresponds to amino acids 61-80.

SEQ ID NO:605 represents the amino acid sequence of Peptide #6 from O8E,which corresponds to amino acids 76-95.

SEQ ID NO:606 represents the amino acid sequence of Peptide #7 from O8E,which corresponds to amino acids 91-110.

SEQ ID NO:607 represents the amino acid sequence of Peptide #8 from O8E,which corresponds to amino acids 106-125.

SEQ ID NO:608 represents the amino acid sequence of Peptide #9 from O8E,which corresponds to amino acids 120-140.

SEQ ID NO:609 represents the amino acid sequence of Peptide #10 fromO8E, which corresponds to amino acids 136-155.

SEQ ID NO:610 represents the amino acid sequence of Peptide #11 fromO8E, which corresponds to amino acids 151-170.

SEQ ID NO:611 represents the amino acid sequence of Peptide #12 fromO8E, which corresponds to amino acids 166-185.

SEQ ID NO:612 represents the amino acid sequence of Peptide #13 fromO8E, which corresponds to amino acids 181-200.

SEQ ID NO:613 represents the amino acid sequence of Peptide #14 fromO8E, which corresponds to amino acids 196-215.

SEQ ID NO:614 represents the amino acid sequence of Peptide #15 fromO8E, which corresponds to amino acids 211-230.

SEQ ID NO:615 represents the amino acid sequence of Peptide #16 fromO8E, which corresponds to amino acids 225-245.

SEQ ID NO:616 represents the amino acid sequence of Peptide #17 fromO8E, which corresponds to amino acids 241-260.

SEQ ID NO:617 represents the amino acid sequence of Peptide #18 fromO8E, which corresponds to amino acids 256-275.

SEQ ID NO:618 represents the amino acid sequence of Peptide #19 fromO8E, which corresponds to amino acids 263-282.

SEQ ID NO:619 is the DNA sequence for the O8E PCR primer, O8E-UP1.

SEQ ID NO:620 is the DNA sequence for the O8E reverse PCR primerdesignated O8E-DN1.

SEQ ID NO:621 is a DNA sequence corresponding to the O8E Rhesusorthologs.

SEQ ID NO:622 is a DNA sequence corresponding to the O8E mouse ortholog.

SEQ ID NO:623 is an amino acid sequence corresponding to the O8E Rhesusorthologs.

SEQ ID NO:624 is an amino acid sequence corresponding to the O8E mouseortholog.

DETAILED DESCRIPTION OF THE INVENTION

U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herern by reference, intheir entirety.

The present invention is directed generally to compositions and theiruse in the therapy and diagnosis of cancer, particularly ovarian cancer.As described further below, illustrative compositions of the presentinvention include, but are not restricted to, polypeptides, particularlyimmunogenic polypeptides, polynucleotides encoding such polypeptides,antibodies and other binding agents, antigen presenting cells (APCs) andimmune system cells (e.g., T cells).

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al., MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Polypeptide Compositions

As used herein, the term “polypeptide” “is used in its conventionalmeaning, i.e., as a sequence of amino acids. The polypeptides are notlimited to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

Particularly illustrative polypeptides of the present invention comprisethose encoded by a polynucleotide sequence set forth in any one of SEQID NOs: 1-311, 313-387, 391, 457, 460-477, 512-570 and 619-622, or asequence that hybridizes under moderately stringent conditions, or,alternatively, under highly stringent conditions, to a polynucleotidesequence set forth in any one of SEQ ID NOs: 1-311, 313-387, 391, 457,460-477, 512-570 and 619-622. Certain other illustrative polypeptides ofthe invention comprise amino acid sequences as set forth in any one ofSEQ ID NOs: 312, 388-389, 392-455, 458-459, 478-511, and 571-618.

The polypeptides of the present invention are sometimes herein referredto as ovarian tumor proteins or ovarian tumor polypeptides, as anindication that their identification has been based at least in partupon their increased levels of expression in ovarian tumor samples.Thus, an “ovarian tumor polypeptide” or “ovarian tumor protein,” refersgenerally to a polypeptide sequence of the present invention, or apolynucleotide sequence encoding such a polypeptide, that is expressedin a substantial proportion of ovarian tumor samples, for examplepreferably greater than about 20%, more preferably greater than about30%, and most preferably greater than about 50% or more of ovarian tumorsamples tested, at a level that is at least two fold, and preferably atleast five fold, greater than the level of expression in normal tissues,as determined using a representative assay provided herein. A ovariantumor polypeptide sequence of the invention, based upon its increasedlevel of expression in tumor cells, has particular utility both as adiagnostic marker as well as a therapeutic target, as further describedbelow.

In certain preferred embodiments, the polypeptides of the invention areimmunogenic, i.e., they react detectably within an immunoassay (such asan ELISA or T-cell stimulation assay) with antisera and/or T-cells froma patient with ovarian cancer. Screening for immunogenic activity can beperformed using techniques well known to the skilled artisan. Forexample, such screens can be performed using methods such as thosedescribed in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988. In one illustrative example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵I-labeled Protein A.

As would be recognized by the skilled artisan, immunogenic portions ofthe polypeptides disclosed herein are also encompassed by the presentinvention. An “immunogenic portion,” as used herein, is a fragment of animmunogenic polypeptide of the invention that itself is immunologicallyreactive (i.e., specifically binds) with the B-cells and/or T-cellsurface antigen receptors that recognize the polypeptide. Immunogenicportions may generally be identified using well known techniques, suchas those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247(Raven Press, 1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

In one preferred embodiment, an immunogenic portion of a polypeptide ofthe present invention is a portion that reacts with antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full-length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Preferably, the level of immunogenic activity of the immunogenicportion is at least about 50%, preferably at least about 70% and mostpreferably greater than about 90% of the immunogenicity for thefull-length polypeptide. In some instances, preferred immunogenicportions will be identified that have a level of immunogenic activitygreater than that of the corresponding full-length polypeptide, e.g.,having greater than about 100% or 150% or more immunogenic activity.

In certain other embodiments, illustrative immunogenic portions mayinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

In another embodiment, a polypeptide composition of the invention mayalso comprise one or more polypeptides that are immunologically reactivewith T cells and/or antibodies generated against a polypeptide of theinvention, particularly a polypeptide having an amino acid sequencedisclosed herein, or to an immunogenic fragment or variant thereof.

In another embodiment of the invention, polypeptides are provided thatcomprise one or more polypeptides that are capable of eliciting T cellsand/or antibodies that are immunologically reactive with one or morepolypeptides described herein, or one or more polypeptides encoded bycontiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

The present invention, in another aspect, provides polypeptide fragmentscomprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous aminoacids, or more, including all intermediate lengths, of a polypeptidecompositions set forth herein, such as those set forth in SEQ ID NOs:312, 388-389, 392-455, 458-459, 478-511, and 571-618, or those encodedby a polynucleotide sequence set forth in a sequence of SEQ ID NOs:1-311, 313-387, 391, 457, 460-477, 512-570 and 619-622.

In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequences set forth herein.

In one preferred embodiment, the polypeptide fragments and variantsprovided by the present invention are immunologically reactive with anantibody and/or T-cell that reacts with a full-length polypeptidespecifically set forth herein.

In another preferred embodiment, the polypeptide fragments and variantsprovided by the present invention exhibit a level of immunogenicactivity of at least about 50%, preferably at least about 70%, and mostpreferably at least about 90% or more of that exhibited by a full-lengthpolypeptide sequence specifically set forth herein.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating their immunogenicactivity as described herein and/or using any of a number of techniqueswell known in the art.

For example, certain illustrative variants of the polypeptides of theinvention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes, pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E. W.and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor11:105 (1971); Saitou, N. Nei, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Add.APL. Math 2:482 (1981), by the identity alignment algorithm of Needlemanand Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similaritymethods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides and polypeptides of theinvention. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. For aminoacid sequences, a scoring matrix can be used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a xenogeneicpolypeptide that comprises an polypeptide having substantial sequenceidentity, as described above, to the human polypeptide (also termedautologous antigen) which served as a reference polypeptide, but whichxenogeneic polypeptide is derived from a different, non-human species.One skilled in the art will recognize that “self” antigens are oftenpoor stimulators of CD8+ and CD4+ T-lymphocyte responses, and thereforeefficient immunotherapeutic strategies directed against tumorpolypeptides require the development of methods to overcome immunetolerance to particular self tumor polypeptides. For example, humansimmunized with prostase protein from a xenogeneic (non human) origin arecapable of mounting an immune response against the counterpart humanprotein, e.g., the human prostase tumor protein present on human tumorcells. Accordingly, the present invention provides methods for purifyingthe xenogeneic form of the tumor proteins set forth herein, such as thepolypeptides set forth in SEQ ID NOs: 312, 388-389, 392-455, 458-459,478-511, and 571-618, or those encoded by polynucleotide sequences setforth in SEQ ID NOs: 1-311, 313-387, 391, 457, 460-477, 512-570 and619-622.

Therefore, one aspect of the present invention provides xenogeneicvariants of the polypeptide compositions described herein. Suchxenogeneic variants generally encompassed by the present invention willtypically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more identity along their lengths, toa polypeptide sequences set forth herein.

More particularly, the invention is directed to mouse, rat, monkey,porcine and other non-human polypeptides which can be used as xenogeneicforms of human polypeptides set forth herein, to induce immune responsesdirected against tumor polypeptides of the invention.

Within other illustrative embodiments, a polypeptide may be a fusionpolypeptide that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the polypeptide or to enable the polypeptide to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of thepolypeptide.

Fusion polypeptides may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion polypeptide isexpressed as a recombinant polypeptide, allowing the production ofincreased levels, relative to a non-fused polypeptide, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46,1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may generally be from 1 to about 50 amino acids in length.Linker sequences are not required when the first and second polypeptideshave non-essential N-terminal amino acid regions that can be used toseparate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described hereintogether with an unrelated immunogenic protein, such as an immunogenicprotein capable of eliciting a recall response. Examples of suchproteins include tetanus, tuberculosis and hepatitis proteins (see, forexample, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences is described in U.S. PatentApplication 60/158,585, the disclosure of which is incorporated hereinby reference in its entirety. Briefly, Ra12 refers to a polynucleotideregion that is a subsequence of a Mycobacterium tuberculosis MTB32Anucleic acid. MTB32A is a serine protease of 32 KD molecular weightencoded by a gene in virulent and avirulent strains of M. tuberculosis.The nucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. 67:3998-4007 (1999), incorporatedherein by reference). C-terminal fragments of the MTB32A coding sequenceexpress at high levels and remain as a soluble polypeptides throughoutthe purification process. Moreover, Ra12 may enhance the immunogenicityof heterologous immunogenic polypeptides with which it is fused. Onepreferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragmentcorresponding to amino acid residues 192 to 323 of MTB32A. Otherpreferred Ra12 polynucleotides generally comprise at least about 15consecutive nucleotides, at least about 30 nucleotides, at least about60 nucleotides, at least about 100 nucleotides, at least about 200nucleotides, or at least about 300 nucleotides that encode a portion ofa Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence(i.e., an endogenous sequence that encodes a Ra12 polypeptide or aportion thereof) or may comprise a variant of such a sequence. Ra12polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions such that the biological activityof the encoded fusion polypeptide is not substantially diminished,relative to a fusion polypeptide comprising a native Ra12 polypeptide.Variants preferably exhibit at least about 70% identity, more preferablyat least about 80% identity and most preferably at least about 90%identity to a polynucleotide sequence that encodes a native Ra12polypeptide or a portion thereof.

Within other preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

Yet another illustrative embodiment involves fusion polypeptides, andthe polynucleotides encoding them, wherein the fusion partner comprisesa targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

Polypeptides of the invention are prepared using any of a variety ofwell known synthetic and/or recombinant techniques, the latter of whichare further described below. Polypeptides, portions and other variantsgenerally less than about 150 amino acids can be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. In one illustrative example, such polypeptides are synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) ofthe invention are isolated. An “isolated” polypeptide is one that isremoved from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

Polynucleotide Compositions The present invention, in other aspects,provides polynucleotide compositions. The terms “DNA” and“polynucleotide” are used essentially interchangeably herein to refer toa DNA molecule that has been isolated free of total genomic DNA of aparticular species. “Isolated,” as used herein, means that apolynucleotide is substantially away from other coding sequences, andthat the DNA molecule does not contain large portions of unrelatedcoding DNA, such as large chromosomal fragments or other functionalgenes or polypeptide coding regions. Of course, this refers to the DNAmolecule as originally isolated, and does not exclude genes or codingregions later added to the segment by the hand of man.

As will be understood by those skilled in the art, the polynucleotidecompositions of this invention can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

As will be also recognized by the skilled artisan, polynucleotides ofthe invention may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. RNA molecules may include HnRNA molecules, which containintrons and correspond to a DNA molecule in a one-to-one manner, andmRNA molecules, which do not contain introns. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a polypeptide/protein of the invention or aportion thereof or may comprise a sequence that encodes a variant orderivative, preferably and immunogenic variant or derivative, of such asequence.

Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth in any one of SEQ ID NOs: 1-311,313-387, 391, 457, 460-477, 512-570 and 619-622, complements of apolynucleotide sequence set forth in any one of SEQ ID NOs: 1-311,313-387, 391, 457, 460-477, 512-570 and 619-622, and degenerate variantsof a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-311,313-387, 391, 457, 460-477, 512-570 and 619-622. In certain preferredembodiments, the polynucleotide sequences set forth herein encodeimmunogenic polypeptides, as described above.

In other related embodiments, the present invention providespolynucleotide variants having substantial identity to the sequencesdisclosed herein in SEQ ID NOs: 1-311, 313-387, 391, 457, 460-477,512-570 and 619-622, for example those comprising at least 70% sequenceidentity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% or higher, sequence identity compared to a polynucleotide sequenceof this invention using the methods described herein, (e.g., BLASTanalysis using standard parameters, as described below). One skilled inthis art will recognize that these values can be appropriately adjustedto determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

In additional embodiments, the present invention provides polynucleotidefragments comprising or consisting of various lengths of contiguousstretches of sequence identical to or complementary to one or more ofthe sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise or consist of at least about10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or morecontiguous nucleotides of one or more of the sequences disclosed hereinas well as all intermediate lengths there between. It will be readilyunderstood that “intermediate lengths”, in this context, means anylength between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22,23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103,etc.; 150, 151, 152, 153, etc.; including all integers through 200-500;500-1,000, and the like. A polynucleotide sequence as described here maybe extended at one or both ends by additional nucleotides not found inthe native sequence. This additional sequence may consist of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesat either end of the disclosed sequence or at both ends of the disclosedsequence.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In certain preferred embodiments, the polynucleotides described above,e.g., polynucleotide variants, fragments and hybridizing sequences,encode polypeptides that are immunologically cross-reactive with apolypeptide sequence specifically set forth herein. In other preferredembodiments, such polynucleotides encode polypeptides that have a levelof immunogenic activity of at least about 50%, preferably at least about70%, and more preferably at least about 90% of that for a polypeptidesequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990);Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E. W.and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Add.APL. Math 2:482 (1981), by the identity alignment algorithm of Needlemanand Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similaritymethods of Pearson and Lipman, Proc. Nat. Acad. Sci. USA 85: 2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants of thepresent invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise or consist of a sequence region ofat least about a 15 nucleotide long contiguous sequence that has thesame sequence as, or is complementary to, a 15 nucleotide longcontiguous sequence disclosed herein will find particular utility.Longer contiguous identical or complementary sequences, e.g., those ofabout 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediatelengths) and even up to full length sequences will also be of use incertain embodiments.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors. For example, one may wish to employ primers fromtowards the termini of the total sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

According to another embodiment of the present invention, polynucleotidecompositions comprising antisense oligonucleotides are provided.Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, provide atherapeutic approach by which a disease can be treated by inhibiting thesynthesis of proteins that contribute to the disease. The efficacy ofantisense oligonucleotides for inhibiting protein synthesis is wellestablished. For example, the synthesis of polygalactauronase and themuscarine type 2 acetylcholine receptor are inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.Nos. 5,739,119 and 5,759,829). Further, examples of antisense inhibitionhave been demonstrated with the nuclear protein cyclin, the multipledrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatalGABA_(A) receptor and human EGF (Jaskulski et al., Science 1988 Jun. 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun. 15;57(2):310-20; U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and5,610,288). Antisense constructs have also been described that inhibitand can be used to treat a variety of abnormal cellular proliferations,e.g., cancer (U.S. Pat. Nos. 5,747,470; 5,591,317 and 5,783,683).

Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein. Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence anddetermination of secondary structure, T_(m), binding energy, andrelative stability. Antisense compositions may be selected based upontheir relative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell. Highly preferred target regions of the mRNA, arethose which are at or near the AUG translation initiation codon, andthose sequences which are substantially complementary to 5′ regions ofthe mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 Jul. 15;25(14):2730-6). It has been demonstrated that several molecules of theMPG peptide coat the antisense oligonucleotides and can be deliveredinto cultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

According to another embodiment of the invention, the polynucleotidecompositions described herein are used in the design and preparation ofribozyme molecules for inhibiting expression of the tumor polypeptidesand proteins of the present invention in tumor cells. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, Proc. Natl. Acad. Sci. USA. 1987December; 84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24;49(2):211-20). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., Cell. 1981 December; 27(3 Pt 2):487-96; Micheland Westhof, J. Mol. Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurekand Shub, Nature. 1992 May 14; 357(6374):173-6). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., Proc. Nat. Acad.Sci. USA. 1992 Aug. 15; 89(16):7305-9). Thus, the specificity of actionof a ribozyme is greater than that of an antisense oligonucleotidebinding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. 1992 Sep. 11; 20(17):4559-65. Examples of hairpin motifs aredescribed by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry 1989 Jun. 13; 28(12):4929-33; Hampel etal., Nucleic Acids Res. 1990 Jan. 25; 18(2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. 1992 Dec. 1; 31(47):11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December; 35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifis described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc. Natl. Acad. Sci. USA,88(19):8826-30 (Oct. 1, 1991); Collins and Olive, Biochemistry32(11):2795-9 (Mar. 23, 1993); and an example of the Group I intron isdescribed in (U.S. Pat. No. 4,987,071). All that is important in anenzymatic nucleic acid molecule of this invention is that it has aspecific substrate binding site which is complementary to one or more ofthe target gene RNA regions, and that it have nucleotide sequenceswithin or surrounding that substrate binding site which impart an RNAcleaving activity to the molecule. Thus the ribozyme constructs need notbe limited to specific motifs mentioned herein.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells Ribozymes expressed from suchpromoters have been shown to function in mammalian cells. Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

In another embodiment of the invention, peptide nucleic acids (PNAs)compositions are provided. PNA is a DNA mimic in which the nucleobasesare attached to a pseudopeptide backbone (Good and Nielsen, AntisenseNucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized ina number methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Areview of PNA including methods of making, characteristics of, andmethods of using, is provided by Corey (Trends Biotechnol 15(6):224-9(June 1997)). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science254(5037):1497-500 (Dec. 6, 1991); Hanvey et al., Science258(5087):1481-5 (Nov. 27, 1992); Hyrup and Nielsen, Bioorg. Med. Chem.4(1):5-23 (January 1996). This chemistry has three importantconsequences: firstly, in contrast to DNA or phosphorothioateoligonucleotides, PNAs are neutral molecules; secondly, PNAs areachiral, which avoids the need to develop a stereoselective synthesis;and thirdly, PNA synthesis uses standard Boc or Fmoc protocols forsolid-phase peptide synthesis, although other methods, including amodified Merrifield method, have been used.

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg. Med. Chem. 3(4):437-45 (April 1995)).The manual protocol lends itself to the production of chemicallymodified PNAs or the simultaneous synthesis of families of closelyrelated PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography, providing yields and purity ofproduct similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem 3(4):437-45 (April 1995); Petersen et al., J PeptSci 1(3):175-83 (May-June 1995); Orum et al., Biotechniques 19(3):472-80(September 1995); Footer et al., Biochemistry. 1996 Aug. 20;35(33):10673-9; Griffith et al., Nucleic Acids Res 23(15):3003-8 (Aug.11, 1995); Pardridge et al., Proc. Natl. Acad. Sci. USA. 92(12):5592-6(Jun. 6, 1995); Boffa et al., Proc. Natl. Acad. Sci. USA. 92(6):1901-5(Mar. 14, 1995); Gambacorti-Passerini et al., Blood 88(4):1411-7 (Aug.15, 1996); Armitage et al., Proc. Natl. Acad. Sci. USA. 94(23):12320-5(Nov. 11, 1997); Seeger et al., Biotechniques 23(3):512-7 (September1997)). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimeric moleculesand their uses in diagnostics, modulating protein in organisms, andtreatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (Anal Chem 65(24):3545-9 (Dec. 15, 1993) and Jensen etal. (Biochemistry. 1997 Apr. 22; 36(16):5072-7). Rose uses capillary gelelectrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparentto the skilled artisan include use in DNA strand invasion, antisenseinhibition, mutational analysis, enhancers of transcription, nucleicacid purification, isolation of transcriptionally active genes, blockingof transcription factor binding, genome cleavage, biosensors, in situhybridization, and the like.

Polynucleotide Identification, Characterization and Expression

Polynucleotides compositions of the present invention may be identified,prepared and/or manipulated using any of a variety of well establishedtechniques (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989, and other like references). For example, a polynucleotidemay be identified, as described in more detail below, by screening amicroarray of cDNAs for tumor-associated expression (i.e., expressionthat is at least two fold greater in a tumor than in normal tissue, asdetermined using a representative assay provided herein). Such screensmay be performed, for example, using the microarray technology ofAffymetrix, Inc. (Santa Clara, Calif.) according to the manufacturer'sinstructions (and essentially as described by Schena et al., Proc. Natl.Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad.Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may beamplified from cDNA prepared from cells expressing the proteinsdescribed herein, such as tumor cells.

Many template dependent processes are available to amplify a targetsequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Any of a number of other template dependent processes, many of which arevariations of the PCR™ amplification technique, are readily known andavailable in the art. Illustratively, some such methods include theligase chain reaction (referred to as LCR), described, for example, inEur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No. 4,883,750; QbetaReplicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880;Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR).Still other amplification methods are described in Great Britain Pat.Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well-known to those of skill in the art.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., atumor cDNA library) using well known techniques. Within such techniques,a library (cDNA or genomic) is screened using one or more polynucleotideprobes or primers suitable for amplification. Preferably, a library issize-selected to include larger molecules. Random primed libraries mayalso be preferred for identifying 5′ and upstream regions of genes.Genomic libraries are preferred for obtaining introns and extending 5′sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, amplification techniques, such as those described above,can be useful for obtaining a full length coding sequence from a partialcDNA sequence. One such amplification technique is inverse PCR (seeTriglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restrictionenzymes to generate a fragment in the known region of the gene. Thefragment is then circularized by intramolecular ligation and used as atemplate for PCR with divergent primers derived from the known region.Within an alternative approach, sequences adjacent to a partial sequencemay be retrieved by amplification with a primer to a linker sequence anda primer specific to a known region. The amplified sequences aretypically subjected to a second round of amplification with the samelinker primer and a second primer specific to the known region. Avariation on this procedure, which employs two primers that initiateextension in opposite directions from the known sequence, is describedin WO 96/38591. Another such technique is known as “rapid amplificationof cDNA ends” or RACE. This technique involves the use of an internalprimer and an external primer, which hybridizes to a polyA region orvector sequence, to identify sequences that are 5′ and 3′ of a knownsequence. Additional techniques include capture PCR (Lagerstrom et al.,PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60, 1991). Other methods employingamplification may also be employed to obtain a full length cDNAsequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GENBANK®. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring.Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thepBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or pSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, any of a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as pBLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.− or aprt.sup.− cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Theuse of visible markers has gained popularity with such markers asanthocyanins, beta-glucuronidase and its substrate GUS, and luciferaseand its substrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Antibody Compositions, Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies and antigen-binding fragmentsthereof, that exhibit immunological binding to a tumor polypeptidedisclosed herein, or to a portion, variant or derivative thereof. Anantibody, or antigen-binding fragment thereof, is said to “specificallybind,” “immunogically bind,” and/or is “immunologically reactive” to apolypeptide of the invention if it reacts at a detectable level (within,for example, an ELISA assay) with the polypeptide, and does not reactdetectably with unrelated polypeptides under similar conditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody refers tothe part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

Binding agents may be further capable of differentiating betweenpatients with and without a cancer, such as ovarian cancer, using therepresentative assays provided herein. For example, antibodies or otherbinding agents that bind to a tumor protein will preferably generate asignal indicating the presence of a cancer in at least about 20% ofpatients with the disease, more preferably at least about 30% ofpatients. Alternatively, or in addition, the antibody will generate anegative signal indicating the absence of the disease in at least about90% of individuals without the cancer. To determine whether a bindingagent satisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. Preferably, a statistically significant number of sampleswith and without the disease will be assayed. Each binding agent shouldsatisfy the above criteria; however, those of ordinary skill in the artwill recognize that binding agents may be used in combination to improvesensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J. Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site whichretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.(1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residueswhich are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein). Solvent accessibilities of V regionamino acids can be deduced from the known three-dimensional structurefor human and murine antibody fragments. There are two general steps inveneering a murine antigen-binding site. Initially, the FRs of thevariable domains of an antibody molecule of interest are compared withcorresponding FR sequences of human variable domains obtained from theabove-identified sources. The most homologous human V regions are thencompared residue by residue to corresponding murine amino acids. Theresidues in the murine FR which differ from the human counterpart arereplaced by the residues present in the human moiety using recombinanttechniques well known in the art. Residue switching is only carried outwith moieties which are at least partially exposed (solvent accessible),and care is exercised in the replacement of amino acid residues whichmay have a significant effect on the tertiary structure of V regiondomains, such as proline, glycine and charged amino acids.

In this manner, the resultant “veneered” murine antigen-binding sitesare thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

In another embodiment of the invention, monoclonal antibodies of thepresent invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

T Cell Compositions

The present invention, in another aspect, provides T cells specific fora tumor polypeptide disclosed herein, or for a variant or derivativethereof. Such cells may generally be prepared in vitro or ex vivo, usingstandard procedures. For example, T cells may be isolated from bonemarrow, peripheral blood, or a fraction of bone marrow or peripheralblood of a patient, using a commercially available cell separationsystem, such as the Isolex™ System, available from Nexell Therapeutics,Inc. (Irvine, Calif.; see also U.S. Pat. Nos. 5,240,856; 5,215,926; WO89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may bederived from related or unrelated humans, non-human mammals, cell linesor cultures.

T cells may be stimulated with a polypeptide, polynucleotide encoding apolypeptide and/or an antigen presenting cell (APC) that expresses sucha polypeptide. Such stimulation is performed under conditions and for atime sufficient to permit the generation of T cells that are specificfor the polypeptide of interest. Preferably, a tumor polypeptide orpolynucleotide of the invention is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the presentinvention if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994. Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a tumor polypeptide (100 ng/ml-100 μg/ml,preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a tumor polypeptide, polynucleotideor polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Tumorpolypeptide-specific T cells may be expanded using standard techniques.Within preferred embodiments, the T cells are derived from a patient, arelated donor or an unrelated donor, and are administered to the patientfollowing stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a tumor polypeptide, polynucleotide or APC can be expandedin number either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a tumor polypeptide, or a short peptidecorresponding to an immunogenic portion of such a polypeptide, with orwithout the addition of T cell growth factors, such as interleukin-2,and/or stimulator cells that synthesize a tumor polypeptide.Alternatively, one or more T cells that proliferate in the presence ofthe tumor polypeptide can be expanded in number by cloning. Methods forcloning cells are well known in the art, and include limiting dilution.

T Cell Receptor Compositions

The T cell receptor (TCR) consists of 2 different, highly variablepolypeptide chains, termed the T-cell receptor α and β chains, that arelinked by a disulfide bond (Janeway, Travers, Walport. Immunobiology.Fourth Ed., 148-159. Elsevier Science Ltd/Garland Publishing. 1999). Theα/β heterodimer complexes with the invariant CD3 chains at the cellmembrane. This complex recognizes specific antigenic peptides bound toMHC molecules. The enormous diversity of TCR specificities is generatedmuch like immunoglobulin diversity, through somatic gene rearrangement.The β chain genes contain over 50 variable (V), 2 diversity (D), over 10joining (J) segments, and 2 constant region segments (C). The α chaingenes contain over 70 V segments, and over 60 J segments but no Dsegments, as well as one C segment. During T cell development in thethymus, the D to J gene rearrangement of the β chain occurs, followed bythe V gene segment rearrangement to the DJ. This functional VDJβ exon istranscribed and spliced to join to a Cβ. For the α chain, a Vα genesegment rearranges to a Jα gene segment to create the functional exonthat is then transcribed and spliced to the Cα. Diversity is furtherincreased during the recombination process by the random addition of Pand N-nucleotides between the V, D, and J segments of the b chain andbetween the V and J segments in the α chain (Janeway, Travers, Walport.Immunobiology. Fourth Ed., 98 and 150. Elsevier Science Ltd/GarlandPublishing. 1999).

The present invention, in another aspect, provides TCRs specific for apolypeptide disclosed herein, or for a variant or derivative thereof. Inaccordance with the present invention, polynucleotide and amino acidsequences are provided for the V-J or V-D-J junctional regions or partsthereof for the alpha and beta chains of the T-cell receptor whichrecognize tumor polypeptides described herein. In general, this aspectof the invention relates to T-cell receptors which recognize or bindtumor polypeptides presented in the context of MHC. In a preferredembodiment the tumor antigens recognized by the T-cell receptorscomprise a polypeptide of the present invention. For example, cDNAencoding a TCR specific for a ovarian tumor peptide can be isolated fromT cells specific for a tumor polypeptide using standard molecularbiological and recombinant DNA techniques.

This invention further includes the T-cell receptors or analogs thereofhaving substantially the same function or activity as the T-cellreceptors of this invention which recognize or bind tumor polypeptides.Such receptors include, but are not limited to, a fragment of thereceptor, or a substitution, addition or deletion mutant of a T-cellreceptor provided herein. This invention also encompasses polypeptidesor peptides that are substantially homologous to the T-cell receptorsprovided herein or that retain substantially the same activity. The term“analog” includes any protein or polypeptide having an amino acidresidue sequence substantially identical to the T-cell receptorsprovided herein in which one or more residues, preferably no more than 5residues, more preferably no more than 25 residues have beenconservatively substituted with a functionally similar residue and whichdisplays the functional aspects of the T-cell receptor as describedherein.

The present invention further provides for suitable mammalian hostcells, for example, non-specific T-cells, that are transfected with apolynucleotide encoding TCRs specific for a polypeptide describedherein, thereby rendering the host cell specific for the polypeptide.The α and β chains of the TCR may be contained on separate expressionvectors or alternatively, on a single expression vector that alsocontains an internal ribosome entry site (IRES) for cap-independenttranslation of the gene downstream of the IRES. Said host cellsexpressing TCRs specific for the polypeptide may be used, for example,for adoptive immunotherapy of ovarian cancer as discussed further below.

In further aspects of the present invention, cloned TCRs specific for apolypeptide recited herein may be used in a kit for the diagnosis ofovarian cancer. For example, the nucleic acid sequence or portionsthereof, of tumor-specific TCRs can be used as probes or primers for thedetection of expression of the rearranged genes encoding the specificTCR in a biological sample. Therefore, the present invention furtherprovides for an assay for detecting messenger RNA or DNA encoding theTCR specific for a polypeptide.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell, TCR, and/orantibody compositions disclosed herein in pharmaceutically-acceptablecarriers for administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will be understood that, if desired, a composition as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceuticalcompositions are provided comprising one or more of the polynucleotide,polypeptide, antibody, TCR, and/or T-cell compositions described hereinin combination with a physiologically acceptable carrier. In certainpreferred embodiments, the pharmaceutical compositions of the inventioncomprise immunogenic polynucleotide and/or polypeptide compositions ofthe invention for use in prophylactic and theraputic vaccineapplications. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Generally,such compositions will comprise one or more polynucleotide and/orpolypeptide compositions of the present invention in combination withone or more immunostimulants.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic compositions, e.g.,vaccine compositions, of the present invention comprise DNA encoding oneor more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the polynucleotide maybe administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

Various adeno-associated virus (MV) vector systems have also beendeveloped for polynucleotide delivery. MV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the polynucleotidesencoding polypeptides of the present invention by gene transfer includethose derived from the pox family of viruses, such as vaccinia virus andavian poxvirus. By way of example, vaccinia virus recombinantsexpressing the novel molecules can be constructed as follows. The DNAencoding a polypeptide is first inserted into an appropriate vector sothat it is adjacent to a vaccinia promoter and flanking vaccinia DNAsequences, such as the sequence encoding thymidine kinase (TK). Thisvector is then used to transfect cells which are simultaneously infectedwith vaccinia. Homologous recombination serves to insert the vacciniapromoter plus the gene encoding the polypeptide of interest into theviral genome. The resulting TK.sup.(−) recombinant can be selected byculturing the cells in the presence of 5-bromodeoxyuridine and pickingviral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Nat. Acad. Sci. USA 87:6743-6747(1990); Fuerst et al., Proc. Natl. Acad. Sci. USA 83:8122-8126 (1986).

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. 268:6866-6869 (1993)and Wagner et al., Proc. Natl. Acad. Sci. USA 89:6099-6103 (1992), canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

In certain embodiments, a polynucleotide may be integrated into thegenome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell, TCR,and/or APC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol® toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformulaHO(CH₂CH₂O)_(n)-A-R,  ( I)wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (ie., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251,1998) and have been shown to be effective as a physiologicaladjuvant for eliciting prophylactic or therapeutic antitumor immunity(see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general,dendritic cells may be identified based on their typical shape (stellatein situ, with marked cytoplasmic processes (dendrites) visible invitro), their ability to take up, process and present antigens with highefficiency and their ability to activate naïve T cell responses.Dendritic cells may, of course, be engineered to express specificcell-surface receptors or ligands that are not commonly found ondendritic cells in vivo or ex vivo, and such modified dendritic cellsare contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowipox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

In another illustrative embodiment, calcium phosphate core particles areemployed as carriers, vaccine adjuvants, or as controlled releasematrices for the compositions of this invention. Exemplary calciumphosphate particles are disclosed, for example, in published patentapplication No. WO/0046147.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature 1997 Mar. 27; 386(6623):410-4; Hwang et al.,Crit Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat. Nos.5,641,515; 5,580,579 and 5,792,451). Tablets, troches, pills, capsulesand the like may also contain any of a variety of additional components,for example, a binder, such as gum tragacanth, acacia, cornstarch, orgelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar, or both. Of course, any material used in preparing any dosageunit form should be pharmaceutically pure and substantially non-toxic inthe amounts employed. In addition, the active compounds may beincorporated into sustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. Nos. 5,543,158; 5,641,515and 5,399,363. In certain embodiments, solutions of the active compoundsas free base or pharmacologically acceptable salts may be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations generally will contain apreservative to prevent the growth of microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212. Likewise,the delivery of drugs using intranasal microparticle resins (Takenaga etal., J Controlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. Nos. 5,567,434; 5,552,157;5,565,213; 5,738,868 and 5,795,587, each specifically incorporatedherein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J. Biol. Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al.,DNA Cell Biol. 1990 April; 9(3):221-9). In addition, liposomes are freeof the DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, he use of liposomes does not appearto be associated with autoimmune responses or unacceptable toxicityafter systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Cancer Therapeutic Methods

Immunologic approaches to cancer therapy are based on the recognitionthat cancer cells can often evade the body's defenses against aberrantor foreign cells and molecules, and that these defenses might betherapeutically stimulated to regain the lost ground, e.g., pgs. 623-648in Klein, Immunology (Wiley-Interscience, New York, 1982). Numerousrecent observations that various immune effectors can directly orindirectly inhibit growth of tumors has led to renewed interest in thisapproach to cancer therapy, e.g., Jager, et al., Oncology 2001;60(1):1-7; Renner, et al., Ann Hematol 2000 December; 79(12):651-9.

Four-basic cell types whose function has been associated with antitumorcell immunity and the elimination of tumor cells from the body are: i)B-lymphocytes which secrete immunoglobulins into the blood plasma foridentifying and labeling the nonself invader cells; ii) monocytes whichsecrete the complement proteins that are responsible for lysing andprocessing the immunoglobulin-coated target invader cells; iii) naturalkiller lymphocytes having two mechanisms for the destruction of tumorcells, antibody-dependent cellular cytotoxicity and natural killing; andiv) T-lymphocytes possessing antigen-specific receptors and having thecapacity to recognize a tumor cell carrying complementary markermolecules (Schreiber, H., 1989, in Fundamental Immunology (ed). W. E.Paul, pp. 923-955).

Cancer immunotherapy generally focuses on inducing humoral immuneresponses, cellular immune responses, or both. Moreover, it is wellestablished that induction of CD4⁺ T helper cells is necessary in orderto secondarily induce either antibodies or cytotoxic CD8⁺ T cells.Polypeptide antigens that are selective or ideally specific for cancercells, particularly ovarian cancer cells, offer a powerful approach forinducing immune responses against ovarian cancer, and are an importantaspect of the present invention.

Therefore, in further aspects of the present invention, thepharmaceutical compositions described herein may be used to stimulate animmune response against cancer, particularly for the immunotherapy ofovarian cancer. Within such methods, the pharmaceutical compositionsdescribed herein are administered to a patient, typically a warm-bloodedanimal, preferably a human. A patient may or may not be afflicted withcancer. Pharmaceutical compositions and vaccines may be administeredeither prior to or following surgical removal of primary tumors and/ortreatment such as administration of radiotherapy or conventionalchemotherapeutic drugs. As discussed above, administration of thepharmaceutical compositions may be by any suitable method, includingadministration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical and oralroutes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Monoclonal antibodies may be labeled with any of a variety of labels fordesired selective usages in detection, diagnostic assays or therapeuticapplications (as described in U.S. Pat. Nos. 6,090,365; 6,015,542;5,843,398; 5,595,721; and 4,708,930, hereby incorporated by reference intheir entirety as if each was incorporated individually). In each case,the binding of the labelled monoclonal antibody to the determinant siteof the antigen will signal detection or delivery of a particulartherapeutic agent to the antigenic determinant on the non-normal cell. Afurther object of this invention is to provide the specific monoclonalantibody suitably labelled for achieving such desired selective usagesthereof.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patients ascompared to non-vaccinated patients. In general, for pharmaceuticalcompositions and vaccines comprising one or more polypeptides, theamount of each polypeptide present in a dose ranges from about 25 μg to5 mg per kg of host. Suitable dose sizes will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which may beperformed using samples obtained from a patient before and aftertreatment.

Cancer Detection and Diagnostic Compositions, Methods and Kits

In general, a cancer may be detected in a patient based on the presenceof one or more ovarian tumor proteins and/or polynucleotides encodingsuch proteins in a biological sample (for example, blood, sera, sputumurine and/or tumor biopsies) obtained from the patient. In other words,such proteins may be used as markers to indicate the presence or absenceof a cancer such as ovarian cancer. In addition, such proteins may beuseful for the detection of other cancers. The binding agents providedherein generally permit detection of the level of antigen that binds tothe agent in the biological sample.

Polynucleotide primers and probes may be used to detect the level ofmRNA encoding a tumor protein, which is also indicative of the presenceor absence of a cancer. In general, a tumor sequence should be presentat a level that is at least two-fold, preferably three-fold, and morepreferably five-fold or higher in tumor tissue than in normal tissue ofthe same type from which the tumor arose. Expression levels of aparticular tumor sequence in tissue types different from that in whichthe tumor arose are irrelevant in certain diagnostic embodiments sincethe presence of tumor cells can be confirmed by observation ofpredetermined differential expression levels, e.g., 2-fold, 5-fold, etc,in tumor tissue to expression levels in normal tissue of the same type.

Other differential expression patterns can be utilized advantageouslyfor diagnostic purposes. For example, in one aspect of the invention,overexpression of a tumor sequence in tumor tissue and normal tissue ofthe same type, but not in other normal tissue types, e.g., PBMCs, can beexploited diagnostically. In this case, the presence of metastatic tumorcells, for example in a sample taken from the circulation or some othertissue site different from that in which the tumor arose, can beidentified and/or confirmed by detecting expression of the tumorsequence in the sample, for example using RT-PCR analysis. In manyinstances, it will be desired to enrich for tumor cells in the sample ofinterest, e.g., PBMCs, using cell capture or other like techniques.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof a cancer in a patient may be determined by (a) contacting abiological sample obtained from a patient with a binding agent; (b)detecting in the sample a level of polypeptide that binds to the bindingagent; and (c) comparing the level of polypeptide with a predeterminedcut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length ovarian tumor proteins and polypeptide portions thereof towhich the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the tumor protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with ovarian least about 95% of thatachieved at equilibrium between bound and unbound polypeptide. Those ofordinary skill in the art will recognize that the time necessary toachieve equilibrium may be readily determined by assaying the level ofbinding that occurs over a period of time. At room temperature, anincubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer, such as ovariancancer, the signal detected from the reporter group that remains boundto the solid support is generally compared to a signal that correspondsto a predetermined cut-off value. In one preferred embodiment, thecut-off value for the detection of a cancer is the average mean signalobtained when the immobilized antibody is incubated with samples frompatients without the cancer. In general, a sample generating a signalthat is three standard deviations above the predetermined cut-off valueis considered positive for the cancer. In an alternate preferredembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive for acancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the tumor proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use tumor polypeptides to detectantibodies that bind to such polypeptides in a biological sample. Thedetection of such tumor protein specific antibodies may correlate withthe presence of a cancer.

A cancer may also, or alternatively, be detected based on the presenceof T cells that specifically react with a tumor protein in a biologicalsample. Within certain methods, a biological sample comprising CD4⁺and/or CD8⁺ T cells isolated from a patient is incubated with a tumorpolypeptide, a polynucleotide encoding such a polypeptide and/or an APCthat expresses at least an immunogenic portion of such a polypeptide,and the presence or absence of specific activation of the T cells isdetected. Suitable biological samples include, but are not limited to,isolated T cells. For example, T cells may be isolated from a patient byroutine techniques (such as by Ficoll/Hypaque density gradientcentrifugation of peripheral blood lymphocytes). T cells may beincubated in vitro for 2-9 days (typically 4 days) at 37° C. withpolypeptide (e.g., 5-25 μg/ml). It may be desirable to incubate anotheraliquot of a T cell sample in the absence of tumor polypeptide to serveas a control. For CD4⁺ T cells, activation is preferably detected byevaluating proliferation of the T cells. For CD8⁺ T cells, activation ispreferably detected by evaluating cytolytic activity. A level ofproliferation that is at least two fold greater and/or a level ofcytolytic activity that is at least 20% greater than in disease-freepatients indicates the presence of a cancer in the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a tumor protein in a biological sample.For example, at least two oligonucleotide primers may be employed in apolymerase chain reaction (PCR) based assay to amplify a portion of atumor cDNA derived from a biological sample, wherein at least one of theoligonucleotide primers is specific for (i.e., hybridizes to) apolynucleotide encoding the tumor protein. The amplified cDNA is thenseparated and detected using techniques well known in the art, such asgel electrophoresis.

Similarly, oligonucleotide probes that specifically hybridize to apolynucleotide encoding a tumor protein may be used in a hybridizationassay to detect the presence of polynucleotide encoding the tumorprotein in a biological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding a tumorprotein of the invention that is at least 10 nucleotides, and preferablyat least 20 nucleotides, in length. Preferably, oligonucleotide primersand/or probes hybridize to a polynucleotide encoding a polypeptidedescribed herein under moderately stringent conditions, as definedabove. Oligonucleotide primers and/or probes which may be usefullyemployed in the diagnostic methods described herein preferably are atleast 10-40 nucleotides in length. In a preferred embodiment, theoligonucleotide primers comprise at least 10 contiguous nucleotides,more preferably at least 15 contiguous nucleotides, of a DNA moleculehaving a sequence as disclosed herein. Techniques for both PCR basedassays and hybridization assays are well known in the art (see, forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263,1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another aspect of the present invention, cell capture technologiesmay be used in conjunction, with, for example, real-time PCR to providea more sensitive tool for detection of metastatic cells expressingovarian tumor antigens. Detection of ovarian cancer cells in biologicalsamples, e.g., bone marrow samples, peripheral blood, and small needleaspiration samples is desirable for diagnosis and prognosis in ovariancancer patients.

Immunomagnetic beads coated with specific monoclonal antibodies tosurface cell markers, or tetrameric antibody complexes, may be used tofirst enrich or positively select cancer cells in a sample. Variouscommercially available kits may be used, including Dynabeads® EpithelialEnrich (Dynal Biotech, Oslo, Norway), StemSep™ (StemCell Technologies,Inc., Vancouver, BC), and RosetteSep (StemCell Technologies). A skilledartisan will recognize that other methodologies and kits may also beused to enrich or positively select desired cell populations. Dynabeads®Epithelial Enrich contains magnetic beads coated with mAbs specific fortwo glycoprotein membrane antigens expressed on normal and neoplasticepithelial tissues. The coated beads may be added to a sample and thesample then applied to a magnet, thereby capturing the cells bound tothe beads. The unwanted cells are washed away and the magneticallyisolated cells eluted from the beads and used in further analyses.

RosetteSep can be used to enrich cells directly from a blood sample andconsists of a cocktail of tetrameric antibodies that targets a varietyof unwanted cells and crosslinks them to glycophorin A on red bloodcells (RBC) present in the sample, forming rosettes. When centrifugedover Ficoll, targeted cells pellet along with the free RBC. Thecombination of antibodies in the depletion cocktail-determines whichcells will be removed and consequently which cells will be recovered.Antibodies that are available include, but are not limited to: CD2, CD3,CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25,CD29, CD33, CD34, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66B,CD66e, HLA-DR, IgE, and TCRαβ.

Additionally, it is contemplated in the present invention that mAbsspecific for ovarian tumor antigens can be generated and used in asimilar manner. For example, mAbs that bind to tumor-specific cellsurface antigens may be conjugated to magnetic beads, or formulated in atetrameric antibody complex, and used to enrich or positively selectmetastatic ovarian tumor cells from a sample. Once a sample is enrichedor positively selected, cells may be lysed and RNA isolated. RNA maythen be subjected to RT-PCR analysis using ovarian tumor-specificprimers in a real-time PCR assay as described herein. One skilled in theart will recognize that enriched or selected populations of cells may beanalyzed by other methods (e.g., in situ hybridization or flowcytometry).

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer may be performed overtime, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple tumor protein markersmay be assayed within a given sample. It will be apparent that bindingagents specific for different proteins provided herein may be combinedwithin a single assay. Further, multiple primers or probes may be usedconcurrently. The selection of tumor protein markers may be based onroutine experiments to determine combinations that results in optimalsensitivity. In addition, or alternatively, assays for tumor proteinsprovided herein may be combined with assays for other known tumorantigens.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a tumor protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a tumor protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a tumor protein.Such an oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding atumor protein.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Identification of Representative Ovarian CarcinomaProtein cDNAs

This Example illustrates the identification of cDNA molecules encodingovarian carcinoma proteins.

Anti-SCID mouse sera (generated against sera from SCID mice carryinglate passage ovarian carcinoma) was pre-cleared of E. coli and phageantigens and used at a 1:200 dilution in a serological expressionscreen. The library screened was made from a SCID-derived human ovariantumor (OV9334) using a directional RH oligo(dT) priming cDNA libraryconstruction kit and the AScreen vector (Novagen). A bacteriophagelambda screen was employed. Approximately 400,000 pfu of the amplifiedOV9334 library were screened.

196 positive clones were isolated. Certain sequences that appear to benovel are provided in FIGS. 1A-1S and SEQ ID NO:1 to 71. Three completeinsert sequences are shown in FIGS. 2A-2C (SEQ ID NO:72 to 74). Otherclones having known sequences are presented in FIGS. 15A-15EEE (SEQ IDNO:82 to 310). Database searches identified the following sequences thatwere substantially identical to the sequences presented in FIGS.15A-15EEE.

These clones were further characterized using microarray technology todetermine mRNA expression levels in a variety of tumor and normaltissues. Such analyses were performed using a Synteni (Palo Alto,Calif.) microarray, according to the manufacturer's instructions. PCRamplification products were arrayed on slides, with each productoccupying a unique location in the array. mRNA was extracted from thetissue sample to be tested, reverse transcribed and fluorescent-labeledcDNA probes were generated. The microarrays were probed with the labeledcDNA probes and the slides were scanned to measure fluorescenceintensity. Data was analyzed using Synteni's provided GEMtools software.The results for one clone (13695, also referred to as O8E) are shown inFIG. 3.

Example 2 Identification of Ovarian Carcinoma cDNAs Using MicroarrayTechnology

This Example illustrates the identification of ovarian carcinomapolynucleotides by PCR subtraction and microarray analysis. Microarraysof cDNAs were analyzed for ovarian tumor-specific expression using aSynteni (Palo Alto, Calif.) microarray, according to the manufacturer'sinstructions (and essentially as described by Schena et al., Proc. Natl.Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad.Sci. USA 94:2150-2155, 1997).

A PCR subtraction was performed using a tester comprising cDNA of fourovarian tumors (three of which were metastatic tumors) and a driver ofcDNA form five normal tissues (adrenal gland, lung, pancreas, spleen andbrain). cDNA fragments recovered from this subtraction were subjected toDNA microarray analysis where the fragments were PCR amplified, adheredto chips and hybridized with fluorescently labeled probes derived frommRNAs of human ovarian tumors and a variety of normal human tissues. Inthis analysis, the slides were scanned and the fluorescence intensitywas measured, and the data were analyzed using Synteni's GEMtoolssoftware. In general, sequences showing at least a 5-fold increase inexpression in tumor cells (relative to normal cells) were consideredovarian tumor antigens. The fluorescent results were analyzed and clonesthat displayed increased expression in ovarian tumors were furthercharacterized by DNA sequencing and database searches to determine thenovelty of the sequences.

Using such assays, an ovarian tumor antigen was identified that is asplice fusion between the human T-cell leukemia virus type I oncoproteinTAX (see Jin et al., Cell 93:81-91, 1998) and an extracellular matrixprotein called osteonectin. A splice junction sequence exists at thefusion point. The sequence of this clone is presented in FIG. 4 and SEQID NO:75. Osteonectin, unspliced and unaltered, was also identified fromsuch assays independently.

Further clones identified by this method are referred to herein as 3f,6b, 8e, 8h, 12c and 12h. Sequences of these clones are shown in FIGS. 5to 9 and SEQ ID NO:76 to 81. Microarray analyses were performed asdescribed above, and are presented in FIGS. 10 to 14. A full lengthsequence encompassing clones 3f, 6b, 8e and 12h was obtained byscreening an ovarian tumor (SCID-derived) cDNA library. This 2996 basepair sequence (designated O772P) is presented in SEQ ID NO:311, and theencoded 914 amino acid protein sequence is shown in SEQ ID NO:312. PSORTanalysis indicates a Type 1a transmembrane protein localized to theplasma membrane.

In addition to certain of the sequences described above, this screenidentified the following sequences which are described in detail inTable 2:

TABLE 2 Sequence Comments OV4vG11 (SEQ ID NO: 313) human clone 1119D9 onchromosome 20p12 OV4vB11 (SEQ ID NO: 314) human UWGC: y14c094 fromchromosome 6p21 OV4vD9 (SEQ ID NO: 315) human clone 1049G16 chromosome20q12-13.2 OV4vD5 (SEQ ID NO: 316) human KIAA0014 gene OV4vC2 (SEQ IDNO: 317) human KIAA0084 gene OV4vF3 (SEQ ID NO: 318) human chromosome 19cosmid R31167 OV4VC1 (SEQ ID NO: 319) Novel OV4vH3 (SEQ ID NO: 320)Novel OV4vD2 (SEQ IDNO: 321) novel O815P (SEQ ID NO: 322) novel OV4vC12(SEQ ID NO: 323) novel OV4vA4 (SEQ ID NO: 324) novel OV4vA3 (SEQ ID NO:325) novel OV4v2A5 (SEQ ID NO: 326) novel O819P (SEQ ID NO: 327) novelO818P (SEQ ID NO: 328) novel O817P (SEQ ID NO: 329) novel O816P (SEQ IDNO: 330) novel Ov4vC5 (SEQ ID NO: 331) novel 21721 (SEQ ID NO: 332)human lumican 21719 (SEQ ID NO: 333) human retinoic acid-binding proteinII 21717 (SEQ ID NO: 334) human26S proteasome ATPase subunit 21654 (SEQID NO: 335) human copine I 21627 (SEQ ID NO: 336) human neuron specificgamma-2 enolase 21623 (SEQ ID NO: 337) human geranylgeranyl transferaseII 21621 (SEQ ID NO: 338) human cyclin-dependent protein kinase 21616(SEQ ID NO: 339) human prepro-megakaryocyte potentiating factor 21612(SEQ ID NO: 340) human UPH1 21558 (SEQ ID NO: 341) human RalGDS-like 2(RGL2) 21555 (SEQ ID NO: 342) human autoantigen P542 21548 (SEQ ID NO:343) human actin-related protein (ARP2) 21462 (SEQ ID NO: 344) humanhuntingtin interacting protein 21441 (SEQ ID NO: 345) human 90K product(tumor associated antigen) 21439 (SEQ ID NO: 346) human guaninenucleotide regulator protein (tim1) 21438 (SEQ ID NO: 347) human Kuautoimmune (p70/p80) antigen 21237 (SEQ ID NO: 348) human S-laminin21436 (SEQ ID NO: 349) human ribophorin I 21435 (SEQ ID NO: 350) humancytoplasmic chaperonin hTRiC5 21425 (SEQ ID NO: 351) humanEMX2 21423(SEQ ID NO: 352) human p87/p89 gene 21419 (SEQ ID NO: 353) humanHPBRII-7 21252 (SEQ ID NO: 354) human T1-227H 21251 (SEQ ID NO: 355)human cullin I 21247 (SEQ ID NO: 356) kunitz type protease inhibitor(KOP) 21244-1 (SEQ ID NO: 357) human protein tyrosine phosphatasereceptor F (PTPRF) 21718 (SEQ ID NO: 358) human LTR repeat OV2-90 (SEQID NO: 359) novel Human zinc finger (SEQ ID NO: 360) Human polyA bindingprotein (SEQ ID NO: 361) Human pleitrophin (SEQ ID NO: 362) Human PACclone 278C19 (SEQ ID NO: 363) Human LLRep3 (SEQ ID NO: 364) Human Kunitztype protease inhib (SEQ ID NO: 365) Human KIAA0106 gene (SEQ ID NO:366) Human keratin (SEQ ID NO: 367) Human HIV-1TAR (SEQ ID NO: 368)Human glia derived nexin (SEQ ID NO: 369) Human fibronectin (SEQ ID NO:370) Human ECMproBM40 (SEQ ID NO: 371) Human collagen (SEQ ID NO: 372)Human alpha enolase (SEQ ID NO: 373) Human aldolase (SEQ ID NO: 374)Human transf growth factor BIG H3 (SEQ ID NO: 375) Human SPARCosteonectin (SEQ ID NO: 376) Human SLP1 leucocyte protease (SEQ ID NO:377) Human mitochondrial ATP synth (SEQ ID NO: 378) Human DNA seq clone461P17 (SEQ ID NO: 379) Human dbpB pro Y box (SEQ ID NO: 380) Human 40kDa keratin (SEQ ID NO: 381) Human arginosuccinate synth (SEQ ID NO:382) Human acidic ribosomal phosphoprotein (SEQ ID NO: 383) Human coloncarcinoma laminin binding pro (SEQ ID NO: 384)

This screen further identified multiple forms of the clone O772P,referred to herein as 21013, 21003 and 21008. PSORT analysis indicatesthat 21003 (SEQ ID NO:386; translated as SEQ ID NO:389) and 21008 (SEQID NO:387; translated as SEQ ID NO:390) represent Type 1a transmembraneprotein forms of O772P. 21013 (SEQ ID NO:385; translated as SEQ IDNO:388) appears to be a truncated form of the protein and is predictedby PSORT analysis to be a secreted protein.

Additional sequence analysis resulted in a full length clone for O8E(2627 bp, which agrees with the message size observed by Northernanalysis; SEQ ID NO:391). This nucleotide sequence was obtained asfollows: the original O8E sequence (OrigO8Econs) was found to overlap by33 nucleotides with a sequence from an EST clone (IMAGE#1987589). Thisclone provided 1042 additional nucleotides upstream of the original O8Esequence. The link between the EST and O8E was confirmed by sequencingmultiple PCR fragments generated from an ovary primary tumor libraryusing primers to the unique EST and the O8E sequence (ESTxO8EPCR). Fulllength status was further indicated when anchored PCR from the ovarytumor library gave several clones (AnchoredPCR cons) that all terminatedupstream of the putative start methionine, but failed to yield anyadditional sequence information. FIG. 16 presents a diagram thatillustrates the location of each partial sequence within the full lengthO8E sequence.

Two protein sequences may be translated from the full length O8E. For“a” (SEQ ID NO:393) begins with a putative start methionine. A secondform “b” (SEQ ID NO:392) includes 27 additional upstream residues to the5′ end of the nucleotide sequence.

Example 3

This example discloses the identification and characterization ofantibody epitopes recognized by the O8E polyclonal anti-sera.

Rabbit anti-sera was raised against E. coli derived O8E recombinantprotein and tested for antibody epitope recognition against 20 or 21 merpeptides that correspond to the O8E amino acid sequence. Peptidesspanning amino acid regions 31 to 65, 76 to 110, 136 to 200 and 226 to245 of the full length O8E protein were recognized by an acid elutedpeak and/or a salt eluted peak from affinity purified anti-O8E sera.Thus, the corresponding amino acid sequences of the above peptidesconstitute the antibody epitopes recognized by affinity purifiedanti-O8E antibodies.

ELISA analysis of anti-O8E rabbit sera is shown in FIG. 23, and ELISAanalysis of affinity purified rabbit anti-O8E polyclonal antibody isshown in FIG. 24.

For epitope mapping, 20 or 21 mer peptides corresponding to the O8Eprotein were synthesized. For antibody affinity purification, rabbitanti-O8E sera was run over an O8E-sepharose column, then antibody waseluted with a salt buffer containing 0.5 M NaCl and 20 mM PO₄, followedby an acid elution step using 0.2 M Glycine, pH 2.3. Purified antibodywas neutralized by the addition of 1M Tris, pH 8 and buffer exchangedinto phosphate buffered saline (PBS). For enzyme linked immunosorbantassay (ELISA) analysis, O8E peptides and O8E recombinant protein werecoated onto 96 well flat bottom plates at 2 μg/ml for 2 hours at roomtemperature (RT). Plates were then washed 5 times with PBS+0.1% Tween 20and blocked with PBS+1% bovine serum albumin (BSA) for 1 hour. Affinitypurified anti-O8E antibody, either an acid or salt eluted fraction, wasthen added to the wells at 1 μg/ml and incubated at RT for 1 hr. Plateswere again washed, followed by the addition of donkeyanti-rabbit-Ig-horseradish peroxidase (HRP) antibody for 1 hour at RT.Plates were washed, then developed by the addition of the chromagenicsubstrate 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (described by Bos etal., J. of Immunoassay 2:187-204 (1981); available from Sigma (St.Louis, MO)). The reaction was incubated 15 minutes at RT and thenstopped by the addition of 1 N H₂SO₄. Plates were read at an opticaldensity of 450 (OD450) in an automated plate reader. The sequences ofpeptides corresponding to the O8E antibody epitopes are disclosed hereinas SEQ ID NO: 394-415. Antibody epitopes recognized by the O8Epolyclonal anti-sera are disclosed herein in FIG. 17.

Example 4

This example discloses IHC analysis of O8E expression in ovarian cancertissue samples.

For immunohistochemistry studies, paraffin-embedded formalin fixedovarian cancer tissue was sliced into 8 micron sections. Steam heatinduced epitope retrieval (SHIER) in 0.1 M sodium citrate buffer (pH6.0) was used for optimal staining conditions. Sections were incubatedwith 10% serum/PBS for 5 minutes. Primary antibody (anti-O8E rabbitaffinity purified polyclonal antibody) was added to each section for 25min followed by a 25 min incubation with an anti-rabbit biotinylatedantibody. Endogenous peroxidase activity was blocked by three 1.5 minincubations with hydrogen peroxidase. The avidin biotin complex/horseradish peroxidase system was used along with DAB chromogen to visualizeantigen expression. Slides were counterstained with hematoxylin. One(papillary serous carcinoma) of six ovarian cancer tissue sectionsdisplayed O8E immunoreactivity. Upon optimization of the stainingconditions, 4/5 ovarian cancer samples stained positive using the O8Epolyclonal antibody. O8E expression was localized to the plasmamembrane.

Six ovarian cancer tissues were analyzed with the anti-O8E rabbitpolyclonal antibody. One (papillary serous carcinoma) of six ovariancancer tissue samples stained positive for O8E expression. O8Eexpression was localized to the surface membrane.

Example 5

This example discloses O8E peptides that are predicted to bind HLA-A2and to be immunogenic for CD8 T cell responses in humans.

Potential HLA-A2 binding peptides of O8E were predicted by using thefull-length open-reading frame (ORF) from O8E and running it through“Episeek,” a program used to predict MHC binding peptides. The programused is based on the algorithm published by Parker, K. C. et al., J.Immunol. 152(1):163-175 (1994) (incorporated by reference herein in itsentirety). 10-mer and 9-mer peptides predicted to bind HLA-0201 aredisclosed herein as SEQ ID NO: 416-435 and SEQ ID NO: 436-455,respectively.

Example 6

This example discloses O8E cell surface expression measured byfluoresence activated cell sorting.

For FACS analysis, cells were washed with ice cold staining buffer(PBS/1% BSA/azide). Next, the cells were incubated for 30 minutes on icewith 10 micrograms/ml of affinity purified rabbit anti-B305D polyclonalantibody. The cells were washed 3 times with staining buffer and thenincubated with a 1:100 dilution of a goat anti-rabbit Ig (H+L)-FITCreagent (Southern Biotechnology) for 30 minutes on ice. Following 3washes, the cells were resuspended in staining buffer containing prodiumiodide, a vital stain that allows for identification of permeable cells,and analyzed by FACS. O8E surface expression was confirmed on SKBR3breast cancer cells and HEK293 cells that stably overexpress the cDNAfor O8E. Neither MB415 cells nor HEK293 cells stably transfected with acontrol irrelevant plasmid DNA showed surface expression of O8E (FIGS.18 and 19).

Example 7

This example further evaluates the expression and surface localizationof O8E.

For expression and purification of antigen used for immunization, O8Eexpressed in an E. coli recombinant expression system was grownovernight in LB Broth with the appropriate antibiotics at 37° C. in ashaking incubator. The next morning, 10 ml of the overnight culture wasadded to 500 ml of 2×YT plus appropriate antibiotics in a 2L-baffledErlenmeyer flask. When the Optical Density (at 560 nanometers) of theculture reached 0.4-0.6 the cells were induced with IPTG (1 mM). 4 hoursafter induction with IPTG the cells were harvested by centrifugation.The cells were then washed with phosphate buffered saline andcentrifuged again. The supernatant was discarded and the cells wereeither frozen for future use or immediately processed. Twentymilliliters of lysis buffer was added to the cell pellets and vortexed.To break open the E. coli cells, this mixture was then run through theFrench Press at a pressure of 16,000 psi. The cells were thencentrifuged again and the supernatant and pellet were checked bySDS-PAGE for the partitioning of the recombinant protein. For proteinthat localized to the cell pellet, the pellet was resuspended in 10 mMTris pH 8.0, 1% CHAPS and the inclusion body pellet was washed andcentrifuged again. This procedure was repeated twice more. The washedinclusion body pellet was solubilized with either 8 M urea or 6 Mguanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. Thesolubilized protein was added to 5 ml of nickel-chelate resin (Qiagen)and incubated for 45 min to 1 hour at room temperature with continuousagitation. After incubation, the resin and protein mixture were pouredthrough a disposable column and the flow through was collected. Thecolumn was then washed with 10-20 column volumes of the solubilizationbuffer. The antigen was then eluted from the column using 8M urea, 10 mMtris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. ASDS-PAGE gel was run to determine which fractions to pool for furtherpurification. As a final purification step, a strong anion exchangeresin such as Hi-Prep Q (Biorad) was equilibrated with the appropriatebuffer and the pooled fractions from above were loaded onto the column.Each antigen was eluted off of the column with an increasing saltgradient. Fractions were collected as the column was run and anotherSDS-PAGE gel was run to determine which fractions from the column topool. The pooled fractions were dialyzed against 10 mM Tris pH 8.0. Thismaterial was then evaluated for acceptable purity as determined bySDS-PAGE or HPLC, concentration as determined by Lowry assay or AminoAcid Analysis, identity as determined by amino terminal proteinsequence, and endotoxin level as determined by the Limulus (LAL) assay.The proteins were then vialed after filtration through a 0.22 micronfilter and the antigens were frozen until needed for immunization.

For generation of polyclonal anti-sera, 400 micrograms of each prostateantigen was combined with 100 micrograms of muramyldipeptide (MDP).Equal volume of Incomplete Freund's Adjuvant (IFA) was added and thenmixed. Every four weeks animals were boosted with 100 micrograms ofantigen mixed with an equal volume of IFA. Seven days following eachboost the animal was bled. Sera was generated by incubating the blood at4° C. for 12-24 hours followed by centrifugation.

For characterization of polyclonal antisera, 96 well plates were coatedwith antigen by incubating with 50 microliters (typically 1 microgram)at 4° C. for 20 hrs. 250 microliters of BSA blocking buffer was added tothe wells and incubated at RT for 2 hrs. Plates were washed 6 times withPBS/0.01% tween. Anti-O8E rabbit sera or affinity purified anti-O8eantibody was diluted in PBS. Fifty microliters of diluted antibody wasadded to each well and incubated at RT for 30 min. Plates were washed asdescribed above before 50 microliters of goat anti-rabbit horse radishperoxidase (HRP) at a 1:10000 dilution was added and incubated at RT for30 min. Plates were washed as described above and 100 microliters of TMBmicrowell Peroxidase Substrate was added to each well. Following a 15minute incubation in the dark at room temperature the calorimetricreaction was stopped with 100 microliters of 1N H2SO4 and readimmediately at 450 nm. All polyclonal antibodies showed immunoreactivityto the O8E antigen.

For recombinant expression in mammalian HEK293 cells, full length O8EcDNA was subcloned into the mammalian expression vectors pcDNA3.1+ andpCEP4 (Invitrogen) which were modified to contain His and FLAG epitopetags, respectively. These constructs were transfected into HEK293 cells(ATCC) using Fugene 6 reagent (Roche). Briefly, HEK293 cells were platedat a density of 100,000 cells/ml in DMEM (Gibco) containing 10% FBS(Hyclone) and grown overnight. The following day, 2 ul of Fugene6 wasadded to 100 ul of DMEM containing no FBS and incubated for 15 minutesat room temperature. The Fugene6/DMEM mixture was then added to 1 ug ofO8E/pCEP4 or O8E/pcDNA3.1 plasmid DNA and incubated for 15 minutes atroom temperature. The Fugene/DNA mix was then added to the HEK293 cellsand incubated for 48-72 hrs at 37° C. with 7% CO2. Cells were rinsedwith PBS then collected and pelleted by centrifugation. For Western blotanalysis, whole cell lysates were generated by incubating the cells inTriton-X100 containing lysis buffer for 30 minutes on ice. Lysates werethen cleared by centrifugation at 10,000 rpm for 5 minutes at 4 C.Samples were diluted with SDS-PAGE loading buffer containingbeta-mercaptoethanol, then boiled for 10 minutes prior to loading theSDS-PAGE gel. Protein was transferred to nitrocellulose and probed usinganti-O8E rabbit polyclonal sera #2333L at a dilution of 1:750. The blotwas revealed with a goat anti-rabbit Ig coupled to HRP followed byincubation in ECL substrate.

For FACS analysis, cells were washed further with ice cold stainingbuffer (PBS+1% BSA+Azide). Next, the cells were incubated for 30 minuteson ice with 10 ug/ml of Protein A purified anti-O8E polyclonal sera. Thecells were washed 3 times with staining buffer and then incubated with a1:100 dilution of a goat anti-rabbit Ig(H+L)-FITC reagent (SouthernBiotechnology) for 30 minutes on ice. Following 3 washes, the cells wereresuspended in staining buffer containing Propidium Iodide (PI), a vitalstain that allows for the identification of permeable cells, andanalyzed by FACS.

From these experiments, the results of which are illustrated in FIGS.20-21, O8E expression was detected on the surface of transfected HEK293cells and SKBR3 cells by FACS analysis using rabbit anti-O8E sera.Expression was also detected in transfected HEK293 cell lysates byWestern blot analysis (FIG. 22).

Example 8 Generation and Characterization of Anti-O8E mAbs

Mouse monoclonal antibodies were raised against E. coli derived O8Eproteins as follows. A/J mice were immunized intraperitoneally (IP) withComplete Freund's Adjuvant (CFA) containing 50 μg recombinant O8E,followed by a subsequent IP boost with Incomplete Freund's Adjuvant(IFA) containing 10 μg recombinant O8E protein. Three days prior toremoval of the spleens, the mice were immunized intravenously withapproximately 50 μg of soluble O8E recombinant protein. The spleen of amouse with a positive titer to O8E was removed, and a single-cellsuspension made and used for fusion to SP2/0 myeloma cells to generate Bcell hybridomas. The supernatants from the hybrid clones were tested byELISA for specificity to recombinant O8E, and epitope mapped usingpeptides that spanned the entire O8E sequence. The mAbs were also testedby flow cytometry for their ability to detect O8E on the surface ofcells stably transfected with O8E and on the surface of a breast tumorcell line.

For ELISA analysis, 96 well plates were coated with either recombinantO8E protein or overlapping 20-mer peptides spanning the entire O8Emolecule at a concentration of either 1-2 μg/ml or 10 μg/ml,respectively. After coating, the plates were washed 5 times with washingbuffer (PBS+0.1% Tween-20) and blocked with PBS containing 0.5% BSA,0.4% Tween-20. Hybrid supernatants or purified mAbs were then added andthe plates incubated for 60 minutes at room temperature. The plates werewashed 5 times with washing buffer and the secondary antibody,donkey-anti mouse Ig linked to horseradish peroxidase (HRP)(JacksonImmunoResearch), was added for 60 minutes. The plates were again washed5 times in washing buffer, followed by the addition of the peroxidasesubstrate. Of the hybridoma clones generated, 15 secreted mAbs thatrecognized the entire O8E protein. Epitope mapping revealed that ofthese 15 clones, 14 secreted mAbs that recognized the O8E amino acidresidues 61-80 and one clone secreted a mAb that recognized amino acidresidues 151-170.

For flow cytometric analysis, HEK293 cells which had been stablytransfected with O8E and SKBR3 cells which express O8E mRNA, wereharvested and washed in flow staining buffer (PBS+1% BSA+Azide). Thecells were incubated with the supernatant from the mAb hybrids for 30minutes on ice followed by 3 washes with staining buffer. The cells wereincubated with goat-anti mouse Ig-FITC for 30 minutes on ice, followedby three washes with staining buffer before being resuspended in washbuffer containing propidium iodide. Flow cytometric analysis revealedthat 15/15 mAbs were able to detect O8E protein expressed on the surfaceof O8E-transfected HEK293 cells. 6/6 mAbs tested on SKBR3 cells wereable to recognize surface expressed O8E.

Example 9 Extended DNA and Protein Sequence Analysis of Sequence O772P

A full-length sequence encompassing clones 3f, 6b, 8e, and 12 wasobtained by screening an ovarian tumor (SCID-derived) cDNA librarydescribed in detail in Example 2. This 2996 base pair sequence,designated O772P, is presented in SEQ ID NO: 311, and the encoded 914amino acid protein sequence is shown in SEQ ID NO: 312. The DNA sequenceO772P was searched against public databases including Genbank and showeda significant hit to Genbank Accession number AK024365 (SEQ ID NO: 457).This Genbank sequence was found to be 3557 base pairs in length andencodes a protein 1156 amino acids in length (SEQ ID NO: 459). Atruncated version of this sequence, residues 25-3471, in which residue25 corresponds to the first ATG initiation codon in the Genbanksequence, (SEQ ID NO: 456), encodes a protein that is 1148 amino acidsin length (SEQ ID NO: 458). The published DNA sequence (SEQ ID NO: 457)differs from O772P in that it has a 5 base pair insertion correspondingto bases 958-962 of SEQ ID NO: 457. This insertion results in a frameshift such that SEQ ID NO: 457 encodes an additional N-terminal proteinsequence relative to O772P (SEQ ID NO: 312). In addition, O772P encodesa unique N-terminal portion contained in residues 1-79 (SEQ ID NO: 460).The N-terminal portion of SEQ ID NO: 456, residues 1-313, also containsunique sequence and is listed as SEQ ID NO: 461.

Example 10 The Generation of Polyclonal Antibodies forImmunohistochemistry and Flow Cytometric Analysis of the Cell AssociatedExpression Pattern of Molecule O772P

The O772P molecule was identified in Examples 2 and 9 of thisapplication. To evaluate the subcellular localization and specificity ofantigen expression in various tissues, polyclonal antibodies weregenerated against O772P. To produce these antibodies, O772P-1 (aminoacids 44-772 of SEQ ID NO:312) and O772P-2 (477-914 of SEQ ID NO:312)were expressed in an E. coli recombinant expression system and grownovernight at 37° C. in LB Broth. The following day, 10 ml of theovernight culture was added to 500 ml of 2×YT containing the appropriateantibiotics. When the optical density of the cultures (560 nanometers)reached 0.4-0.6 the cells were induced with IPTG. Following induction,the cells were harvested, washed, lysed and run through a French Pressat a pressure of 16000 psi. The cells were then centrifuged and thepellet checked by SDS-PAGE for the partitioning of the recombinantprotein. For proteins that localize to the cell pellet, the pellet wasresuspended in 10 mM Tris, pH 8.0, 1% CHAPS and the inclusion bodypellet washed and centrifuged. The washed inclusion body was solubilizedwith either 8M urea or 6M guanidine HCL containing 10 mM Tris, pH 8.0,plus 10 mM imidazole. The solubilized protein was then added to 5 ml ofnickel-chelate resin (Qiagen) and incubated for 45 minutes at roomtemperature.

Following the incubation, the resin and protein mixture was pouredthrough a column and the flow through collected. The column was washedwith 10-20 column volumes of buffer and the antigen eluted using 8Murea, 10 mM Tris, pH 8.0, and 300 mM imidazole and collected in 3 mlfractions. SDS-PAGE was run to determine which fractions to pool forfurther purification. As a final purification step, a strong anionexchange resin was equilibrated with the appropriate buffer and thepooled fractions were loaded onto the column. Each antigen was elutedfrom the column with an increasing salt gradient. Fractions werecollected and analyzed by a SDS-PAGE to determine which fractions fromthe column to pool. The pooled fractions were dialyzed against 10 mMTris, pH 8.0, and the resulting protein was submitted for qualitycontrol for final release. The release criteria were: (a) purity asdetermined by SDS-PAGE or HPLC, (b) concentration as determined by Lowryassay or Amino Acid Analysis, (c) identity as determined by aminoterminal protein, and (d) endotoxin levels as determined by the Limulus(LAL) assay. The proteins were then filtered through a 0.22 μM filterand frozen until needed for immunizations.

To generate polyclonal antisera, 400 μg of O772P-1 or O772P-2 wascombined with 100 μg of muramyldipeptide (MDP). The rabbits wereimmunized every 4 weeks with 100 μg of antigen mixed with an equalvolume of Incomplete Freund's Adjuvant (IFA). Seven days following eachboost, the animals were bled and sera was generated by incubating theblood at 4° C. for 12-24 hours followed by centrifugation.

To characterize the antisera, 96 well plates were coated with antigenfollowed by blocking with BSA. Rabbit sera was diluted in PBS and addedto each well. The plates were then washed, and goat anti-rabbithorseradish peroxidase (HRP). The plates were again washed and TMBmicrowell Peroxidase Substrate was added. Following this incubation, thecolormetric reaction was stopped and the plates read immediately at 450nm. All polyclonal antibodies showed immunoreactivity to the appropriateantigen.

Immunohistochemistry analysis of O772P expression was performed onparaffin-embedded formalin fixed tissue. O772P was found to be expressedin normal ovary and ovarian tumor, but not in normal heart, kidney,colon, lung or liver. Additionally, immunohistochemistry and flowcytometric analysis indicates that O772P is a plasma membrane-associatedmolecule. O772P contains 1 plasma transmembrane domain predicted to beencoded by amino acids 859-880. The N-terminus of O772P is extracellularand is encoded by amino acids 1-859, while the C-terminus isintracellular. Sequence analysis shows that there are 17 potentialN-linked glycosylation sites.

Example 11 O772P is Expressed on the Surface of Primary Ovarian TumorCells

For recombinant expression in mammalian cells, the O772P-21008 (SEQ IDNO:387) and O772P full length cDNA (SEQ ID NO:311 encoding the proteinof SEQ ID NO:312) were subcloned into mammalian expression vectors pBIBor pCEP4 respectively. These constructs were transfected into HEK293cells using Fugene 6 (Roche). The HEK cells were then plated at adensity of 100,000 cells/ml in DMEM containing fetal bovine serum (FBS)and grown overnight. The following day, 2 μl of Fugene 6 was added to100 μl of DMEM, which contained no FBS, and incubated for 15 minutes atroom temperature. The Fugene 6/DMEM mixture was then added to 1 μg ofO772P/pBIB or O772P/pCEP4 plasmid DNA and incubated for an additional 15minutes at room temperature. The Fugene 6/DNA mix was then added to theHEK293 cells and incubated for 48-72 hours at 37° C. with 7% CO₂. Thecells were rinsed and pelleted by centrifugation.

For Western Blot analysis, whole cell lysates were generated byincubating the cells in lysis buffer followed by clarification bycentrifugation. The samples were diluted and run on SDS-PAGE. The gelwas then transferred to nitrocellulose and probed using purifiedanti-O772P-2 rabbit polyclonal antibody. The blot was revealed with agoat anti-rabbit Ig coupled to HRP followed by incubation in ECLsubstrate. Western Blot analysis revealed that O772P-21008 could bedetected in HEK293 cells that had been transfected with O772P.

To determine the cell expression profile of O772P in cells, primaryovarian tumor cells were grown in SCID mice. The cells were retrievedfrom the mice and analyzed by flow cytometry. Briefly, cells washed incold staining buffer containing PBS, 1% BSA, and Na Azide. The cellswere incubated for 30 minutes with 10 μg/ml of purified anti-O772P-1 andO772P-2 polyclonal sera. Following this incubation, the cells werewashed three times in staining buffer and incubated with goatanti-rabbit Ig (H+L) conjugated to FITC (Southern Biotechnology). Thecells were washed and resuspended in staining buffer containingPropidium Iodide (PI), a vital stain that identifies non-viable cells.The cells were then analyzed using Fluorescence Activated Cell Sorting(FACS). FACS analysis revealed that O772P was present on the cellssurface. Surface expression of O772P on tumor cells allows for immunetargeting by therapeutic antibodies.

Example 12 Functional Characterization of Anti-O8E Monoclonal Antibodies

Mouse monoclonal antibodies (mAb) raised against E. coli derived O8E, asdescribed in Example 8, were tested for their ability to promote O8Eantigen internalization. Internalization of the antibody was determinedusing an in vitro cytotoxicity assay. Briefly, HEK293 and O8E/HEKtransfected cells were plated into 96 well plates containing DME plus10% heat-inactivated FBS in the presence of 50 ng/well of purifiedanti-O8E or control antibodies. The isotype of the anti-O8E mabs are asfollows: 11A6-IgG1/kappa, 15C6-IgG2b/kappa, 18A8-IgG2b/kappa, and14F1-IgG2a/kappa. W6/32 is a pan anti-human MHC class I mouse monoclonalantibody that serves as a positive control, and two irrelevant mAbs,Ir-Pharm and Ir-Crxa were included as negative controls. Followingincubation with the O8E specific antibodies or the relevant controlsantibodies, the mAb-zap, a goat anti-mouse Ig-saporin conjugatedsecondary antibody (Advanced Targeting Systems) was added at aconcentration of 100 ng/ml to half of the wells, and the plates wereincubated for 48 to 72 hours at 37° C. in a 7% CO₂ incubator. This assaytakes advantage of the toxic nature of saporin, a ribozyme inactivatingprotein, which when internalized has a cytotoxic effect. Followingincubation with the mAb-zap, internalization was quantitated by theaddition of MTS reagent, followed by reading the OD490 of the plate on amicroplate ELISA reader. FIG. 25 depicts the results from these assays.The top panel represents HEK cells that have not been transfected withO8E and therefore O8E antibody should not bind and be internalized.Levels of proliferation were the same in all samples whether they wereincubated with or without the mAb-zap, with the exception of thepositive control Ab, W6/32. The lower panel represents cells that havebeen transfected with O8E and therefore should bind O8E specificantibodies. Antibodies from the hybridomas 11H6, 14F1, and 15C6, whichrecognize the amino acids 61-80 of O8E were able to promoteinternalization of the O8E surface protein as measured by decreasedlevels of proliferation due to the toxic nature of the mAb-zap (See FIG.25). The antibody generated by the hybridoma 18A8, which recognizesamino acids 151-170 of O8E, was unable to promote internalization asdetermined by normal levels of proliferation either in the absence orpresence of the mAb-zap.

Example 13 Characterization of the Ovarian Tumor Antigen, O772P

The cDNA and protein sequences for multiple forms of the ovarian tumorantigen O772P have been described in the above (e.g., Examples 2 and 9).A Genbank search indicated that O772P has a high degree of similaritywith FLJ14303 (Accession # AK024365; SEQ ID NO:457 and 463). Proteinsequences corresponding to O772P and FLJ14303 are disclosed in SEQ IDNO:478 and 479, respectively. FLJ14303 was identical to the majority ofO772P, with much of the 3′-end showing 100% homology. However, the5′-end of FLJ14303 was found to extend further 5′ than O772P. Inaddition, FLJ14303 contained a 5 bp insert (SEQ ID NO:457) resulting ina frame shift of the amino-terminus protein sequence such that FLJ14303utilizes a different starting methionine than O772P and thereforeencodes a different protein. This insertion was present in the genomicsequence and seen in all EST clones that showed identity to this region,suggesting that FLJ14303 (SEQ ID NO:457) represents a splice variant ofO772P, with an ORF that contains an extended and differentamino-terminus. The additional 5′-nucleotide sequence included repeatsequences that were identified during the genomic mapping of O772P. The5′-end of O772P and the corresponding region of FLJ14303 showed between90-100% homology. Taken together, this suggests that O772P and FLJ14303are different splice variants of the same gene, with different uniquerepeat sequences being spliced into the 5′-end of the gene.

The identification of an additional ten or more repeat sequences withinthe same region of chromosome 19, indicates that there may be many formsof O772P, each with a different 5′-end, due to differential splicing ofdifferent repeat sequences. Northern blot analysis of O772P demonstratedmultiple O772P-hybridizing transcripts of different sizes, some inexcess 10 kb.

Upon further analysis, 13 additional O772P-related sequences wereidentified, the cDNA and amino acid sequences of which are described inTable 3.

TABLE 3 SEQ ID NO: Description Transmembrane Domains 464 LS #1043400.1(cDNA) nd 465 LS #1043400.10 (cDNA) 0 466 LS #1043400.11 (cDNA) 2 467 LS#1043400.12 (cDNA) 2 468 LS #1043400.2 (cDNA) nd 469 LS #1043400.3(cDNA) 470 LS #1043400.5 (cDNA) nd 471 LS #1043400.8 (cDNA) 1 472 LS#1043400.9 (cDNA) 0 473 LS #1043400.6 (cDNA) nd 474 LS #1043400.7 (cDNA)nd 475 LS #1043400.4 (cDNA) nd 476 LS #1397610.1 (cDNA) 0 477 1043400.10Novel 5′ (cDNA) — 480 LS #1043400.9 (amino acid) — 481 LS #1043400.8B(amino acid) — Contains a transmembrane domain 482 LS #1043400.8A (aminoacid) — 483 LS #1043400.12 (amino acid) — Contains a transmembranedomain 484 LS #1043400.11B (amino acid) — Contains a transmembranedomain 485 LS #1043400.11A (amino acid) — 486 LS #1043400.10 (aminoacid) — 487 LS #1043400.1 (amino acid) — nd = not determined

Initially it appeared that these sequences represented overlappingand/or discrete sequences of O772P splice forms that were capable ofencoding polypeptides unique to the specific splice forms of O772P.However, nucleotide alignment of these sequences failed to identify anyidentical regions within the repeat elements. This indicates that thesequences may represent different specific regions of a single O772Pgene, one that contains 16 or more repeat domains, all of which form asingle linear transcript. The 5′-end of sequence LS #1043400.10 (Table2; SEQ ID NO:465) is unique to both O772P and FLJ14303 and contains norepeat elements, indicating that this sequence may represent the 5′-endof O772P.

Previously, transmembrane prediction analysis had indicated that O772Pcontained between 1 and 3 transmembrane spanning domains. This wasverified by the use of immunohistochemistry and flow cytometry, whichdemonstrated the existence of a plasma membrane-associated moleculerepresenting O772P. However, immunohistochemistry also indicated thepresence of secreted form(s) of O772P, possibly resulting from analternative splice form of O772P or from a post-translational cleavageevent. Analysis of several of the sequences presented in Table 2 showedthat sequences 1043400B.12, 1043400.8B, and 1043400.11B all containedtransmembrane regions, while 1043400.8A, 1043400.10, 1043400.1,1043400.11A, and 1043400.9 were all lacking transmembrane sequences,suggesting that these proteins may be secreted.

Analysis indicates a part of O772P is expressed and/or retained on theplasma membrane, making O772P an attractive target for directingspecific immunotherapies, e.g., therapeutic antibodies, against thisprotein. The predicted extracellular domain of O772P is disclosed in SEQID NO:489 and secretion of O772P is likely to occur as a result of acleavage event within the sequence:

SLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQP.

Proteolytic cleavage is most likely to occur at the Lysine (K) atposition 10 of SEQ ID NO:489. The extracellular, transmembrane, andcytoplasmic regions of O772P are all disclosed in SEQ ID NO:488:

Extracellular: SLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYFPNRNEPLTGNSDLPF Transmembrane: WAVILIGLAGLLGLITCLICGVLVTTCytoplasmic: RRRKKEGEYNVQQQCPGYYQSHLDLEDLQ

Example 14 Immunohistochemistry (IHC) Analysis of O8E Expression inOvarian Cancer and Normal Tissues

In order to determine which tissues express the ovarian cancer antigenO8E, IHC analysis was performed on a diverse range of tissue sectionsusing both polyclonal and monoclonal antibodies specific for O8E. Thegeneration of O8E specific polyclonal antibodies is described in detailin Example 8. The monoclonal antibodies used for staining were 11A6 and14F1, both of which are specific for amino acids 61-80 of O8E and 18A8,which recognizes amino acids 151-170 of O8E (see Example 12 for detailson generation).

To perform staining, tissue samples were fixed in formalin solution for12-24 hours and embedded in paraffin before being sliced into 8 micronsections. Steam heat induced epitope retrieval (SHEIR) in 0.1M sodiumcitrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Primaryantibody was then added to each section for 25 minutes followed by 25minutes of incubation with either anti-rabbit or anti-mouse biotinylatedantibody. Endogenous peroxidase activity was blocked by three 1.5 minuteincubations with hydrogen peroxidase. The avidin biotin complex/horseradish peroxidase (ABC/HRP) system was used along with DAB chromogen tovisualize the antigen expression. Slides were counterstained withhematoxylin to visualize the cell nuclei.

Results using rabbit affinity purified polyclonal antibody to O8E (a.a.29-283; for details on the generation of this Ab, see Example 3) arepresented in Table 3. Results using the three monoclonal antibodies arepresented in Table 4.

TABLE 4 Immunohistochemistry analysis of O8E using polyclonal antibodiesTissue O8E Expression Ovarian Cancer Positive Breast Cancer PositiveNormal Ovary Positive Normal Breast Positive Blood Vessel PositiveKidney Negative Lung Negative Colon Negative Liver Negative HeartNegative

TABLE 5 Immunohistochemistry analysis of O8E using monoclonal antibodies11A6 18A8 14F1 Normal Endo- Epi- Endo- Epi- Endo- Tissue thelial thelialthelial thelial thelial Epithelial Skin 2 2 0 0 1 1 Skin 1 1 0 0 1 1Breast 0 1 n/a n/a 1 1 Colon 0 0 0 0 0 0 Jejunum 0 0 0 0 0 0 Colon 0 0 00 0 0 Colon 0 0 0 0 0 0 Ovary 0 0 0 0 1 0 Colon 0 0 0 0 0 1 Liver 0 0 00 1 2 Skin 0 0 0 0 1 0 Duodenum 0 0 0 0 0 0 and Pancreas Appendix 0 0 00 0 0 Ileum 0 0 0 0 0 0 0 = no staining, 1 = light staining, 2 =moderate staining, n/a = not available

Example 15 Epitope Mapping of O772P Polyclonal Antibodies

To perform epitope mapping of O772P, peptides were generated, thesequences of which were derived from the sequence of O772P. Thesepeptides were 15 mers that overlapped by 5 amino acids and weregenerated via chemical synthesis on membrane supports. The peptides werecovalently bound to Whatman 50 cellulose support by their C-terminuswith the N-terminus unbound. In order to determine epitope specificity,the membranes were wet with 100% ethanol for 1 minute, and then blockedfor 16 hours in TBS/Tween/Triton buffer (50 mM Tris, 137 mM NaCl, 2.7 mMKCl, 0.5% BSA, 0.05% Tween 20, 0.05% Triton X-100, pH 7.5). The peptideswere then probed with 2 O772P specific antibodies, O772P-1 (amino acids44-772 of SEQ ID NO:312) and O772P-2 (477-914 of SEQ ID NO:312; seeExample 10 for details of antibody generation), as well as irrelevantrabbit antibodies for controls. The antibodies were diluted to 1 μg/mland incubated with the membranes for 2 hours at room temperature. Themembranes were then washed for 30 minutes in TBS/Tween/Triton buffer,prior to being incubated with a 1:10,000 dilution of HRP-conjugatedanti-rabbit secondary antibody for 2 hours. The membranes were againwashed for 30 minutes in TBS/Tween/Triton and anti-peptide reactivitywas visualized using ECL. Specific epitope binding specificity for eachof the O772P-polyclonal antibodies is described in Table 6.

TABLE 6 SEQ ID NO: Peptide # Anti-O772P1 Anti-O772P2 Peptide Sequence490 2 *** — TCGMRRTCSTLAPGS 491 6 * */— CRLTLLRPEKDGTAT 492 7 * —DGTATGVDAICTHHP 493 8 — — CTHHPDPKSPRLDRE 494 9 *** *** RLDREQLYWELSQLT495 11 */— — LGPYALDNDSLFVNG 496 13 **** — SVSTTSTPGTPTYVL 497 22 — —LRPEKDGEATGVDAI 498 24 ** */— DPTGPGLDREQLYLE 499 27 */— —LDRDSLYVNGFTHRS 500 40 */— — GPYSLDKDSLYLNGY 501 41 — — YLNGYNEPGPDEPPT502 47 *** *** ATFNSTEGVLQHLLR 503 50 — *** QLISLRPEKDGAATG 504 51 — **GAATGVDTTCTYHPD 505 52 — */— TYHPDPVGPGLDIQQ 506 53 — * LDIQQLYWELSQLTH507 58 — * HIVNWNLSNPDPTSS 508 59 — * DPTSSEYITLLRDIQ 509 60 — *LRDIQDKVTTLYKGS 510 61 — *** LYKGSQLHDTFRFCL 511 71 — **DKAQPGTTNYQRNKR * = relative reactive level, —; no binding, ****;maximal binding

Example 16 Identification of a Novel N-Terminal Repeat StructureAssociated with O772P

Various O772P cDNA and protein forms have been identified andcharacterized as detailed above (e.g., Examples 1, 2, 9, and 14).Importantly, O772P RNA and protein have been demonstrated to beover-expressed in ovarian cancer tissue relative to normal tissues andthus represents an attractive target for ovarian cancer diagnostic andtherapeutic applications.

Using bioinformatic analysis of open reading frames (ORFs) from genomicnucleotide sequence identified previously as having homology with O772P,multiple nucleotide repeat sequences were identified in the 5′ region ofthe gene encoding the O772P protein. A number of these repeat sequenceswere confirmed by RT-PCR using primers specific for the individualrepeats. Fragments which contained multiple repeats were amplified fromcDNA, thus confirming the presence of specific repeats and allowing anorder of these repeats to be established.

Unexpectedly, when various sets of O772P sequences derived fromdifferent database and laboratory sources were analyzed, at least 20different repeat structures, each having substantial levels of identitywith each other (see Table 6), were identified in the 5′ region of theO772P gene and the corresponding N-terminal region of the O772P protein.Each repeat comprises a contiguous open reading frame encoding apolypeptide unit that is capable of being spliced to one or more otherrepeats such that concatomers of the repeats are formed in differingnumbers and orders. Interestingly, other molecules have been describedin the scientific literature that have repeating structural domainsanalogous to those described herein for O772P. For example, the mucinfamily of proteins, which are the major glycoprotein component of themucous which coats the surfaces of cells lining the respiratory,digestive and urogenital tracts, have been shown to be composed oftandemly repeated sequences that vary in number, length and amino acidsequence from one mucin to another (Perez-Vilar and Hill, J. Biol. Chem.274(45):31751-31754, 1999).

The various identified repeat structures set forth herein are expectedto give rise to multiple forms of O772P, most likely by alternativesplicing. The cDNA sequences of the identified repeats are set forth inSEQ ID NOs:513-540, 542-546, and 548-567. The encoded amino acidsequences of the repeats are set forth in SEQ ID NOs:574-593. In manyinstances these amino acid sequences represent consensus sequences thatwere derived from the alignment of more than one experimentally derivedsequence.

Each of these splice forms is capable of encoding a unique O772P proteinwith multiple repeat domains attached to a constant carboxy terminalprotein portion of O772P that contains a trans membrane region. The cDNAsequence of the O772P constant region is set forth in SEQ ID NO:568 andthe encoded amino acid sequence is set forth in SEQ ID NO:594.

All of the available O772P sequences that were obtained were broken downinto their identifiable repeats and these sequences were compared usingthe Clustal method with weighted residue weight table (MegAlign softwarewithin DNASTAR sequence analysis package) to identify the relationshipbetween the repeat sequences. Using this information, the ordering dataprovided by the RT-PCR, and sequence alignments (automatic and manual)using SeqMan (DNASTAR), one illustrative consensus full length O772Pcontig was identified comprising 20 distinct repeat units. The cDNA forthis O772P cDNA contig is set forth in SEQ ID NO:569 and the encodedamino acid sequence is set forth in SEQ ID NO:595. This form of theO772P protein includes the following consensus repeat structures in thefollowing order:

SEQ ID NO:572-SEQ ID NO:574-SEQ ID NO:575-SEQ ID NO:576-SEQ IDNO:577-SEQ ID NO:578-SEQ ID NO:579-SEQ ID NO:580-SEQ ID NO:581-SEQ IDNO:582-SEQ ID NO:583-SEQ ID NO:584-SEQ ID NO:585-SEQ ID NO:586-SEQ IDNO:587-SEQ ID NO:588-SEQ ID NO:589-SEQ ID NO:590-SEQ ID NO:591-SEQ IDNO:592-SEQ ID NO:593.

SEQ ID NO:595, therefore, represents one illustrative full-lengthconsensus sequence for the O772P protein. As discussed above, however,based on current knowledge of this protein and based upon scientificliterature describing proteins containing analogous repeatingstructures, many other forms of O772P are expected to exist with eithermore or less repeats. In addition, many forms of O772P are expected tohave differing arrangements, e.g., different orders, of these N-terminalrepeat structures. The existence of multiple forms of O772P havingdiffering numbers of repeats is supported by Northern analysis of O772P.In this study, Northern hybridization of a O772P-specific probe resultedin a smear of multiple O772P-hybridizing transcripts, some in excess 10kb.

Thus, the variable repeat region of the O772 protein can beillustratively represented by the structure Xn—Y, wherein X comprises arepeat structure having at least 50% identity with the consensus repeatsequence set forth in SEQ ID NO:596; n is the number of repeats presentin the protein and is expected to typically be a integer from 1 to about35; Y comprise the O772P constant region sequence set forth in SEQ IDNO:594 or sequences having at least 80% identity with SEQ ID NO:594.Each X present in the Xn repeat region of the O772 molecule isdifferent.

To determine the consensus sequences of each of the 20 repeat regions,sequences that were experimentally determined for a discrete repeatregion were aligned and a consensus sequence determined. In addition todetermining the consensus sequences for individual repeat regions, aconsensus repeat sequence was also determined. This sequence wasobtained by aligning the 20 individual consensus sequences. Variabilityof the repeats was determined by aligning the consensus amino acidsequences from each of the individual repeat regions with the over allrepeat consensus sequence. Identity data is presented in Table 6.

TABLE 7 Percent identities of Repeat Sequences with Reference to theConsensus Repeat Sequence Repeat Number Percent Identity to Consensus(amino acid) SEQ ID NO: Repeat Sequence 2 574 88 3 575 84 4 576 88 5 57789 6 578 93 7 579 90 8 580 91 9 581 88 10 582 85 11 583 86 12 584 87 13585 87 14 586 89 15 587 89 16 588 89 17 589 83 18 590 84 19 591 83 20592 57 21 593 68

Example 17 Generation and Characterization of Anti-O772P MonoclonalAntibodies

Monoclonal antibodies were generated against the O772P-2 protein,specifically amino acid residues 447-914 of SEQ ID NO:312. To producethese antibodies, A/J mice were immunized i.p. with 50 μg of recombinantO772P-2 (rO772P-2) mixed with Complete Freud's Adjuvant, followed by asecond immunization with 10 μg of rO772P-2 in Incomplete Freud'sAdjuvant. Three days prior to the animals being sacrificed, the micewere immunized i.v. with 50 μg of soluble rO772P-2 protein. The spleensfrom mice with positive titers of O772P-2 were harvested, and a singlecell suspension was made and used for fusion to SP2/0 myeloma cells togenerate B cell hybridomas.

The supernatants from the resulting hybrid clones were tested by ELISAfor specificity to the rO772P-2 protein. Briefly, 96 well plates werecoated with rO772P-2 or with one of three 30 mer peptides, whichcorresponded to the extracellualr domain of the O772P protein. Sequencescorresponding to these peptides are disclosed in SEQ ID NO:597-599. TherO772P protein or peptides were coated at a concentration of 1-2 μg/mland 10 μg/ml, respectively, and allowed to incubate for 60 minutes atroom temperature. Following coating, the plates were washed five timeswith PBS containing 0.1% Tween-20, and then blocked with PBS containing0.5% BSA and 0.4% Tween-20 for 1-2 hours at room temperature. Followingthe addition of the hybridoma supernatants the plates were againincubated for 60 minutes at room temperature. The plates were thenwashed as above and donkey-anti-mouse Ig-HRP linked secondary antibody(Jackson ImmunoResearch) was added and incubated for 60 minutes at roomtemperature, followed by a final wash. TMB peroxidase substrate was thenadded and incubated for 5-15 minutes at room temperature in the dark.The reaction was stopped by the addition of 1N H₂SO₄ and the opticaldensity was read at 450 nm. Epitope mapping using ELISA revealed thatthere was 1 hybridoma that recognized peptide #1 (SEQ ID NO:597).

The hybrid supernatants were also tested using flow cytometry todetermine if they were capable of recognizing surface expressed epitopesof the O772P protein. Briefly, transiently transfected O772P-08/HEK293,O772P-3rpt/HEK293, O772P-7rpt/HEK293 cells were harvested and washed,followed by incubation with the hybridoma supernatants on ice for 30minutes. The cells were then washed in staining buffer (1×PBS, 0.5% BSAand 0.01% sodium azide), followed by incubation with goat-anti-mouseIg-FITC for 30 minutes on ice. The cells were again washed, andresuspended in staining buffer containing 1% propidium iodide (todetermine cell viability). The cells were then analyzed for surfaceexpression of the mAbs. Flow cytometry analysis revealed that the 3 ofthe hybridomas tested were able to detect O772P protein expressed in thesurface of O772P-transfected HEK293 cells.

Example 18 Characterization of Human Anti-O8E Monoclonal Antibodies

Monoclonal antibodies (mAb) were generated against the O8E protein, theamino acid sequence of which is disclosed in SEQ ID NO:392. To producethese mAbs, Medarex mice were i.p. immunized repeatedly with multipleforms if recombinant O8E antigens, including O8E protein produced usingE. coli, O8E plasmid DNA and O8E expressing CHO-1 cells. Spleens frommice with positive titers to O8E were collected and used for fusion tomyeloma cells in order to generate B cell hybrids. The supernatants fromthe resulting hybrids were tested by ELISA for specificity to O8Erecombinant protein as well as being tested by flow cytometry forrecognition of surface expressed epitopes of the O8E protein and ininternalization assays.

For ELISA analysis, 96 well plates were coated with recombinant O8E(rO8E) protein or with pools of 5 over-lapping 20-mer peptides that spanthe entire O8E molecule (peptides 1-19, the sequences of which aredisclosed in SEQ ID NOs:600-618). Recombinant proteins and peptides werecoated at a concentration of 1-2 μg/ml and 10 μg/ml, respectively, andthen allowed to incubate for 60 minutes at room temperature. Aftercoating, the plates were washed five times with PBS containing 0.1%Tween-20 and then blocked with PBS containing 0.5% BSA and 0.4% Tween-20for 1-2 hours at room temperature. Following the addition of thehybridoma supernatants, the plates were incubated for 60 minutes at roomtemperature. The plates were washed as above and mouse anti-humanIgG-HRP linked secondary antibody was added and incubated for 60 minutesat room temperature, followed by a final washing as above. TMBperoxidase substrate was added and incubated for 5-15 minutes at roomtemperature in the dark. The reaction was stopped by the addition of 1NH₂SO₄ and the OD was read at 450 nm. Hybridoma 7F5 was shown to reactwith peptides 11-15, corresponding to amino acids 151-230. No otherhybridoma demonstrated reactivity against the O8E specific peptides.Results from this study are presented in Table 7, column 2.

For flow cytometric analysis, HEK293 cells stably transfected with O8Eor SKBR3 cells, which naturally express O8E, were harvested and washed,then incubated with the hybridoma supernatants for 30 minutes on ice.Following this incubation, the cells were washed with staining buffer(1×PBS, 0.5% BSA, and 0.01% sodium azide). Next, mouse anti-humankappa-FITC was added to the cells and allowed to incubate for 30 minuteson ice. Again, the cells were washed followed by resuspension in washbuffer containing 1% propidium iodide. The cells were then subjected toflow cytometric analysis. Results from these experiments are presentedin Table 8, column 3.

For internalization assays, 1×10³ SKBR3 cells/well were plated into 96well plates containing DME plus 10% heat inactivated fetal bovine serumin the presence of 50 ng/well of human anti-O8E hybridoma supernatantsor a control antibody. A mouse anti-human Ig-saporin conjugatedsecondary antibody was then added at various concentrations to thewells, and the plates were incubated for 4 days at 37° C. in a 7% CO₂incubator. To measure any decreases in the amount of proliferation MTS(20 μl/ml: Promega) was added to the cells for 1 to 2 hours.Proliferation was then measured by reading the OD490 of the plate. Themajority of the hybridomas tested were internalized resulting in adecrease in the amount of proliferation detected. The results from thesestudies are summarized in Table 8, column 6. These findings indicatethat the hybridomas can be used to deliver toxins, either directly orindirectly conjugated to the anti-O8E antibodies, to cells that expressO8E on their cell surface. This allows for the specific targeting anddeath of cells that express the ovarian tumor antigen, O8E.

TABLE 8 Summary of anti-O8E hybridoma data ELISA HEK O8E/HEK SKBR3 Clone(O8E Ag) Facs* (MFI) (MFI) % death 7H4 − + 2.29 96.08 50 9C10 − + 2.85105.98 40 12B9 − + 2.22 113.63 12 6F9 − + 4.78 100.36 25 11C11 − nt 2.4426.64 20 2A7 + + 2.52 153.61 25 5A7 + + 2.45 22.76 25 14D2 − + 3.9255.45 15 12B10 − + 2.21 23.74 40 12E.1 + + 2.27 10.87 50 6E.2 − + 2.4531.89 12 2G6 − + 2.43 86.7 30.7 3C7 − + 2.61 112.2 5.6 3D8 − + 2.47105.02 24.2 4B8 − + 2.74 128 29.6 4G5 − + 2.64 68.56 14.1 8D5 − + 2.55131.99 8.2 8G9 − + 2.64 123.4 18 9A5 − + 2.43 89.43 12.3 9B6 − + 2.51175.81 16.3 14A4 − + 2.52 197.33 4.5 18H6 − + 2.58 90.29 −16.2 19C9 − +2.44 47.11 −14.1 20E8 − + 2.51 111.2 −14.9 21B9 − + 2.46 137.36 −2.222G5 − + 2.88 54.66 16.3 23F9 − + 2.53 30.02 13.5 24C2 − + 2.59 57.9 242A7.G4 + + 2.45 42.87 40.5 2A7.G7 + + 2.48 47.28 46.4 2A7.H5 + + 2.4743.57 37 2A7.H8 + + 2.42 45.76 40 2A7.D12 + + 2.67 51.83 44.3 1E9 − +2.67 26.14 68.5 2A2 − + 2.59 156.82 49.4 2D4 − + 2.56 115.79 50 2E4 − +2.54 32.5 58.1 2F9 − + 2.46 92.78 59.5 4C7 − + nt nt nt 5C6 − + 2.5488.19 64.2 5H5 − + 2.66 100.82 52 6F1 − + 2.45 57.81 49.8 7H12 − + 2.56172 45 8A10 − + 2.41 16.47 58.3 8B7 − + nt nt nt 9E11 − + nt nt nt 10D4− + nt nt nt 10G7 − + 2.5  3.04 59.1 11H8 − + 2.43 5.22 9 13A4 − + 2.4553.58 37.5 MFI = mean fluorescence intensity; Facs = fluorescenceactivated cells sorter Internalization assay - 1% death = average OD (+)toxin/average OD (−) toxins × 100

Example 19 Immunohistochemical Analysis Ovarian Cancer Using HumanAnti-O8E Monoclonal Antibodies

Anti-O8E immunoreactivity was tested in breast cancer, ovarian cancerand normal tissues. In order to perform this analysis, paraffin embeddedformalin fixed tissues were sliced into 8 micron sections. Steam heatinduced epitope retrieval (SHIER) in 0.1 M sodium, citrate buffer (pH6.0) was used for optimal staining conditions. Sections were incubatedin PBS containing 10% serum for 5 minutes. Primary O8E mAb (described indetail in Example 18) was then added to each section for 25 minutesfollowed by a 25 minute incubation with an anti-mouse biotinylatedantibody. Endogenous peroxidase was blocked by three 1.5 minuteincubations with hydrogen peroxidase. The avidin biotin complex/horseradish peroxidase (ABC/HRP) system was used along with DAB chromogen tovisualize antigen expression. Slides were counterstained withhematoxylin.

O8E expression was detected in the majority of ovarian tumor samples butnot in normal ovary. O8E expression was also observed in breast cancersamples and at very low levels in normal breast. Of the normal tissuestested (blood vessel, heart, liver, kidney, colon, stomach, skin andlung), only stomach and skin tested positive.

Example 20 Expression of O8E Orthologs

The identification and characterization of related forms of the ovarianspecific antigen O8E has been described above, one representative DNAsequence for which is set forth in SEQ ID NO:391, encoding amino acidsequences set forth in SEQ ID NOs:392-393. O8E is a plasma membraneassociated protein that is over-expressed in ovarian and other cancers.In this example, O8E orthologs from monkey and mouse are identified.

For the cloning of the Rhesus monkey O8E, a PCR primer set was designedusing the human O8E open reading frame sequence. The O8E forward primer,designated O8E-UP1, 5′CAGMGCTTATGGCTTCCCTGGGGCAGACT-3′ (SEQ ID NO:619)corresponds to the first 21 nucleotides of the human O8E ORF includingthe start codon, with a HindIII restriction site added at the 5′-end.The O8E reverse primer, designated O8E-DN1,5′CAGCGGCCGCTTATTTTAGCATCAGGTAAGG-3′ (SEQ ID NO:620) corresponds to thelast 21 nucleotides of the human O8E ORF including the stop codon, witha NotI restriction site added to the 5′-end. The monkey O8E was thenamplified from Rhesus monkey placenta cDNA sample using theabove-described primer set and cloned into pCEP4 mammalian expressionvector. Sequence analysis showed that the O8E sequence derived from theRhesus (rhO8E) cDNA demonstrated 97.9% identity at the cDNA level and98.2% identity at the protein level when compared to human O8E.Representative protein and cDNA sequences are disclosed in SEQ ID NOs:623 and 621, respectively.

For the cloning of the mouse O8E cDNA, the moue EST database wassearched using the human O8E sequence. This search resulted in theidentification of several mouse ESTs that shared greater than 80%identity to the human O8E cDNA sequence. Three of the mouse EST cloneswere obtained and sequenced. One clone, 557246, (Genebank Accessionnumber AA117088) contained the full-length cDNA insert for the mouse O8Ein a mammalian expression vector, pCMV-SPORT2. Sequence analysis showedthat the mouse O8E (rmO8E) shared 82.7% identity at the cDNA level and86.6% identity at the protein level with human O8E. Representativeprotein and cDNA sequences are disclosed in SEQ ID NOs:624 and 622,respectively.

Example 21 Characterization of the Expression Profile of O8E Orthologs

This example demonstrates that mAbs generated against human O8E werecapable of recognizing cells transfected with either Rhesus O8E or mouseO8E.

For expression of the O8E orthologs, HEK293 cells were plated at adensity of 250,000 cells/well in DMEM containing 10% FBS and incubatedfor 4 hours. At the end of the incubation period, 2 μl of Lipofectamine2000 (Invitrogen) was added to 50 μl of Optimen 1 (Invitrogen)containing no FBS and incubated for 5 minutes at room temperature. In adifferent tube 50 μl of Optimen 1 was mixed with ˜1.0 μg of pCEP4vector, pCEP/rhesus monkey O8E, or pCMV-Spert2/mouse O8E plasmid DNA andthe mixture was transferred to the Lipofectamine 2000/Optimen mix. Thecombined mixture was incubated for 20 minutes at room temperature andthen transferred to the HEK293 cells containing 2 ml of fresh DMEMcontaining 10% FBS. The transformed HEK293 cells were incubated forapproximately 72 hours at 37° C. with 7% CO₂.

For Fluorescence Activated Cell Sorting (FACS) analysis, cells werecollected and washed with ice cold staining buffer (PBS containing 1%BSA and Azide). HEK293/human O8E stable transfectants were used as apositive control for the FACS analysis. The cells were incubated for 45minutes at room temperature with human mAbs generated against the humanO8E protein (identified in Example 18) then washed 2 times with stainingbuffer followed by incubation with 20 μl of anti-human IgG-FITC reagent(Pharminigen) for 30 minutes ay room temperature. Following 2 washes,the cells were resuspended in staining buffer containing PropidiumIodide (PI), a vital stain that allows for identification of dead cells,and analyzed by FACS.

Cells transfected with human O8E, monkey O8E and mouse O8E demonstratedO8E specific staining with all human O8E mAbs tested. The results ofthis study are summarized in Table 9.

TABLE 9 Summary of O8E Staining Using Cells Expressing Human O8E, MouseO8E, and Rhesus O8E Orthologs Geometric Mean Fluorescent Intensity(GeoMFI) HEK293- HEK293- HEK293- HEK293- Human mAb pCEP4 huO8E rmO8EmO8E Human IgG 24.45 24.88 38.96 49.65 (control) 1G11 26.40 275.60301.06 300.97 2A7 26.35 419.46 474.83 448.04 2D4 28.57 352.71 416.72436.11 2F9 28.51 381.29 446.81 426.47 5A4 27.08 77.78 72.64 141.51 5C631.47 395.61 464.40 377.79 5H5 32.58 220.14 302.87 323.77 6F1 27.01214.72 211.47 157.42 6F9 29.22 241.65 272.48 212.39 7H12 27.48 365.52416.14 403.88 7H4 27.58 279.54 333.76 305.97 8A10 29.59 371.75 438.83402.82 9B6 26.04 267.77 442.83 415.75 9C10 28.34 260.90 273.98 113.1912B10 25.38 199.61 258.99 99.53 12B9 23.26 272.40 284.54 116.58 12E125.74 302.00 68.24 47.49 13A12 27.03 172.35 196.80 99.19 13D8 25.03386.35 462.70 430.94 14A4 27.45 408.81 471.49 440.18 14D2 27.58 243.74295.29 212.84 15C8 30.12 392.47 448.20 440.61 18H6 26.27 160.84 222.63213.72

Example 22 Analysis of cDNA Expression Using Real-Time PCR

Real-time PCR (see Gibson et al., Genome Research 6:995-1001, 1996; Heidet al., Genome Research 6:986-994, 1996) is a technique that evaluatesthe level of PCR product accumulation during amplification. Thistechnique permits quantitative evaluation of mRNA levels in multiplesamples. Briefly, mRNA is extracted from tumor and normal tissue andcDNA is prepared using standard techniques. Real-time PCR is performed,for example, using a Perkin Elmer/Applied Biosystems (Foster City,Calif.) 7700 Prism instrument. Matching primers and fluorescent probesare designed for genes of interest using, for example, the primerexpress program provided by Perkin Elmer/Applied Biosystems (FosterCity, Calif.). Optimal concentrations of primers and probes areinitially determined by those of ordinary skill in the art, and control(e.g., β-actin) primers and probes are obtained commercially from, forexample, Perkin Elmer/Applied Biosystems (Foster City, Calif.). Toquantitate the amount of specific RNA in a sample, a standard curve isgenerated using a plasmid containing the gene of interest. Standardcurves are generated using the Ct values determined in the real-timePCR, which are related to the initial cDNA concentration used in theassay. Standard dilutions ranging from 10-10⁶ copies of the gene ofinterest are generally sufficient. In addition, a standard curve isgenerated for the control sequence. This permits standardization ofinitial RNA content of a tissue sample to the amount of control forcomparison purposes.

An alternative real-time PCR procedure can be carried out as follows:The first-strand cDNA to be used in the quantitative real-time PCR issynthesized from 20 μg of total RNA that is first treated with DNase I(e.g., Amplification Grade, Gibco BRL Life Technology, Gaitherburg,Md.), using Superscript Reverse Transcriptase (RT) (e.g., Gibco BRL LifeTechnology, Gaitherburg, Md.). Real-time PCR is performed, for example,with a GeneAmp™ 5700 sequence detection system (PE Biosystems, FosterCity, Calif.). The 5700 system uses SYBR™ green, a fluorescent dye thatonly intercalates into double stranded DNA, and a set of gene-specificforward and reverse primers. The increase in fluorescence is monitoredduring the whole amplification process. The optimal concentration ofprimers is determined using a checkerboard approach and a pool of cDNAsfrom ovarian tumors is used in this process. The PCR reaction isperformed in 25 μl volumes that include 2.5 μl of SYBR green buffer, 2μl of cDNA template and 2.5 μl each of the forward and reverse primersfor the gene of interest. The cDNAs used for RT reactions are dilutedapproximately 1:10 for each gene of interest and 1:100 for the β-actincontrol. In order to quantitate the amount of specific cDNA (and henceinitial mRNA) in the sample, a standard curve is generated for each runusing the plasmid DNA containing the gene of interest. Standard curvesare generated using the Ct values determined in the real-time PCR whichare related to the initial cDNA concentration used in the assay.Standard dilution ranging from 20-2×10⁶ copies of the gene of interestare used for this purpose. In addition, a standard curve is generatedfor β-actin ranging from 200 fg-2000 fg. This enables standardization ofthe initial RNA content of a tissue sample to the amount of β-actin forcomparison purposes. The mean copy number for each group of tissuestested is normalized to a constant amount of β-actin, allowing theevaluation of the over-expression levels seen with each of the genes.

Example 23 Peptide Priming of T-Helper Lines

Generation of CD4⁺ T helper lines and identification of peptide epitopesderived from tumor-specific antigens that are capable of beingrecognized by CD4⁺ T cells in the context of HLA class II molecules, iscarried out as follows:

Fifteen-mer peptides overlapping by 10 amino acids, derived from atumor-specific antigen, are generated using standard procedures.Dendritic cells (DC) are derived from PBMC of a normal donor usingGM-CSF and IL-4 by standard protocols. CD4⁺ T cells are generated fromthe same donor as the DC using MACS beads (Miltenyi Biotec, Auburn,Calif.) and negative selection. DC are pulsed overnight with pools ofthe 15-mer peptides, with each peptide at a final concentration of 0.25μg/ml. Pulsed DC are washed and plated at 1×10⁴ cells/well of 96-wellV-bottom plates and purified CD4⁺ T cells are added at 1×10⁵/well.Cultures are supplemented with 60 ng/ml IL-6 and 10 ng/ml IL-12 andincubated at 37° C. Cultures are restimulated as above on a weekly basisusing DC generated and pulsed as above as antigen presenting cells,supplemented with 5 ng/ml IL-7 and 10 U/ml IL-2. Following 4 in vitrostimulation cycles, resulting CD4⁺ T cell lines (each line correspondingto one well) are tested for specific proliferation and cytokineproduction in response to the stimulating pools of peptide with anirrelevant pool of peptides used as a control.

Example 24 Generation of Tumor-Specific CTL Lines Using In VitroWhole-Gene Priming

Using in vitro whole-gene priming with tumor antigen-vaccinia infectedDC (see, for example, Yee et al, The Journal of Immunology,157(9):4079-86, 1996), human CTL lines are derived that specificallyrecognize autologous fibroblasts transduced with a specific tumorantigen, as determined by interferon-γ ELISPOT analysis. Specifically,dendritic cells (DC) are differentiated from monocyte cultures derivedfrom PBMC of normal human donors by growing for five days in RPMI mediumcontaining 10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml humanIL-4. Following culture, DC are infected overnight with tumorantigen-recombinant vaccinia virus at a multiplicity of infection(M.O.I) of five, and matured overnight by the addition of 3 μg/ml CD40ligand. Virus is then inactivated by UV irradiation. CD8+T cells areisolated using a magnetic bead system, and priming cultures areinitiated using standard culture techniques. Cultures are restimulatedevery 7-10 days using autologous primary fibroblasts retrovirallytransduced with previously identified tumor antigens. Following fourstimulation cycles, CD8+T cell lines are identified that specificallyproduce interferon-γ when stimulated with tumor antigen-transducedautologous fibroblasts. Using a panel of HLA-mismatched B-LCL linestransduced with a vector expressing a tumor antigen, and measuringinterferon-γ production by the CTL lines in an ELISPOT assay, the HLArestriction of the CTL lines is determined.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated antibody or antigen binding fragment thereof, whichspecifically binds to the O8E polypeptide of SEQ ID NO: 392 at an aminoacid sequence selected from the group consisting of SEQ ID NOs: 396-400,403-406, 409 and 413-415.
 2. An isolated antibody or antigen bindingfragment thereof, which specifically binds to the O8E polypeptide of SEQID NO: 392 at an amino acid sequence selected from the group consistingof SEQ ID NOs: 398, 414 and
 415. 3. An antibody or antigen bindingfragment of any one of claims 1 or 2, wherein the antibody or antigenbinding fragment is conjugated to a toxin.
 4. An antibody or antigenbinding fragment of claim 3, wherein the toxin is selected from thegroup consisting of a ricin toxin, abrin toxin, diptheria toxin, choleratoxin, gelonin toxin, Pseudomonas exotoxin, Shigella toxin, and pokeweedantiviral protein.
 5. An antibody or antigen binding fragment of any oneof claims 1 or 2, wherein the antibody or antigen binding fragment is ahuman antibody or antigen binding fragment.
 6. A composition comprisingat least one antibody or antigen binding fragment of any one of claims 1or 2 and a physiologically acceptable carrier.
 7. A diagnostic kitcomprising at least one antibody or antigen binding fragment of any oneof claims 1 or 2 and a detection reagent.
 8. A diagnostic kit of claim7, wherein the detection reagent comprises a reporter group.
 9. A methodfor making an antibody comprising immunizing a mammal with a peptidehaving an amino acid sequence selected from any one of SEQ ID NOs:396-400, 403-406, 409 and 413-415.